Cloning of mammalian genes in microbial organisms and methods for pharmacological screening

ABSTRACT

A method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) enocded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins.

BACKGROUND OF THE INVENTION

[0001] Presently, there are several methods available for cloning mammalian genes. The standard approach to cloning mammalian genes requires obtaining purified protein, determining a partial amino acid sequence of the purified protein, using the partial amino acid sequence to produce degenerate oligonucleotide probes, and screening cDNA libraries with these probes in order to obtain cDNA encoding the protein. This method is time consuming and, because of the degeneracy of the probes used, may identify sequences other than those encoding the protein(s) of interest. Many mammalian genes have been cloned this way, including the cGMP phosphodiesterase expressed in retina (Ovchinnikov, Y -A. et al., FEB 223: 169 (1987)).

[0002] A second approach to cloning genes encoding a protein of interest is to use a known gene as a probe to find homologs. This approach is particularly useful when members of a gene family or families are sufficiently homologous. It is reasonable to expect that members of a given gene family can be so cloned once one member of the family has been cloned. The D. melanogaster dunce phosphodiesterase gene was used, for example to clone rat homologs. (Davis, R. L. et al., Proc. Natl. Acad. Sci. USA 86: 3604 (1989); Swinnen, J. V. et al., Proc. Natl. Acad. Sci. USA 86: 5325 (1989)). Although members of one family of phosphodiesterase genes might be cloned once a member of that family has been cloned, it is unclear whether the nucleotide sequences of genes belonging to different phosphodiesterase gene families exhibit sufficient homology to use probes derived from one family to identify members of another family.

[0003] It would be useful to have a method which could be used to clone genes which does not have the limitations of presently available techniques.

SUMMARY OF THE INVENTION

[0004] The present invention relates to a method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which, encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) encoded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins. As described herein, the present method has been used to identify novel mammalian genes which encode cAMP phosphodiesterases and proteins which interact with RAS proteins. These genes, and others that can be derived by the claimed method, are part of this invention, as are the proteins which they encode.

[0005] The present invention further relates to a method of identifying agents which alter (i.e., reduce or stimulate) the activity of the protein products of such mammalian genes expressed in microorganisms, such as yeast. Identification of such agents can be carried out using two types of screening procedures: one based on biochemical assays of mammalian proteins of known enzymatic function and one based on phenotypic assays for proteins of unknown function. In the former case, if the encoded proteins are cAMP phosphodiesterases, pharmacological screens include the assay for agents which alter (i.e., reduce or stimulate) phosphodiesterase activity. In the latter case, if the encoded proteins interact with RAS proteins, pharmacological screens include the assay for agents which reduce or stimulate interactions with RAS proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 is a flow diagram of the steps of the present method of cloning a mammalian gene which encodes a product capable of correcting a genetic alteration in a microorganism which is associated with an identifiable phenotypic characteristic.

[0007]FIG. 2 is a schematic representation of the yeast expression vectors used to clone mammalian cDNAs. FIG. 2A is a schematic representation of yeast expression vector pADNS. FIG. 2B is a schematic representation of yeast expression vector pADANs. The origins of replication (ori, 2μ) and selectable markers (AmpR, LEU2) are shown, as are the alcohol dehydrogenase (ADH) promoter and terminator sequences. The polylinker restriction endonuclease sites are as shown. In FIG. 2B, the stippled area indicates a portion of the ADH coding sequences.

[0008]FIG. 3 is the nucleotide sequence of DPD cDNA (top line) and its deduced amino acid sequence (bottom line). Nucleotide and amino acid coordinates are given in the left hand margin.

[0009]FIG. 4 is the nucleotide sequence and the deduced amino acid sequence of cDNA clone #44. Nucleotide and amino acid coordinates are given in the left hand margin.

[0010]FIG. 5 is the nucleotide sequence and the deduced amino acid sequence of cDNA clone #99. Nucleotide and amino acid coordinates are given in the left hand margin.

[0011]FIG. 6 is the nucleotide and the deduced amino acid sequence of cDNA clone #265. Nucleotide and amino acid coordinates are given in the left hand margin.

[0012]FIG. 7 is the nucleotide sequence and the deduced amino acid sequence of cDNA clone #310. Nucleotide and amino acid coordinates are given in the left hand margin.

[0013]FIG. 8 shows suppression of heat shock resistance resulting from treatment of yeast cultures with a pharmacological agent. Cells on the right plate were pretreated with 100 μM rolipram prior to heat shock treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to a method of cloning a mammalian gene which, when expressed in a microorganism, can modify, complement or suppress, in the microorganism, a genetic alteration or defect which is associated with an identifiable phenotype. Cloning of a selected mammalian gene is carried out according to the present method by introducing the gene into a genetically altered microorganism which has an identifiable phenotypic alteration or characteristic associated with the genetic alteration; maintaining the genetically altered microorganism in which the gene is present under conditions appropriate for cell growth; and selecting cells in which the phenotypic alteration or characteristic is modified as a result of correction, complementation or suppression of the genetic alteration or defect by the introduced mammalian gene. The present invention further relates to mammalian genes cloned by the present method, products (e.g., RNA, proteins) encoded by such genes and antibodies specific for encoded proteins. In particular, the present invention relates to a method of cloning and isolating a selected mammalian gene which, when expressed in yeast having a genetic alteration or defect associated with an identifiable phenotypic characteristic or alteration, corrects, complements or supplements the genetic alteration and modifies or corrects the associated phenotypic characteristic.

[0015] Through use of the method described herein, mammalian genes capable of modifying phenotypic alterations in yeast associated with activation or attenuation of biochemical pathways in which cAMP participates or with biochemical pathways which interact with or are controlled by RAS proteins have been identified, cloned and characterized. As described herein, the subject method has been used to clone mammalian genes encoding cAMP phosphodiesterases or proteins which interact with a RAS protein. This has been accomplished using yeast cells in which there is a genetic alteration in the RAS2 gene and which, as a result, are heat shock sensitive. Additional mammalian genes having these same characteristics can be identified, cloned and characterized using the method described. In each case, an appropriately-selected genetically altered host cell (e.g., yeast) is used for expression of the selected mammalian gene, which generally is introduced as one component of a gene library. The genetic alteration in the host cell is selected in such a manner that it is associated with an identifiable phenotypic characteristic which is corrected upon expression of the mammalian gene. The genetic alteration can be a deletion of a gene or a portion of a gene, a change in nucleotide sequence or any other nucleotide manipulation which renders a gene unable to function normally. The genetic alteration is selected in such a manner that a gene of interest can be identified when it is expressed in the altered host cell.

[0016] The mammalian genes, when expressed in yeast containing a genetic defect in a biochemical pathway, can correct, complement or alter the genetic defect and correct the phenotypic alteration (i.e., produce a phenotype more like that of normal or unaltered yeast). Cells containing the mammalian gene to be cloned are identified on the basis of correction or suppression of the phenotypic characteristic. The correction or suppression need not be complete.

[0017] As described herein, it is now possible to identify inhibitors and activators of cAMP phosphodiesterases, which can be used therapeutically to control or regulate cAMP levels or activity. In addition, it is now possible to identify agents which inhibit or stimulate interaction of gene products with RAS proteins.

[0018] It is one of the objects of this invention to discover, isolate and characterize new genes encoding cAMP phosphodiesterases. It is a further object of this invention to describe methods for identifying chemical agents which inhibit or stimulate cAMP phosphodiesterases and can be used for therapeutic purposes. Both of these objectives can be achieved through use of methods, described herein, for cloning genes encoding cAMP phosphodiesterases and expressing the proteins that they encode in cells with little or no other cAMP phosphodiesterase activity. Typically, cells used for expressing the proteins to be analyzed for cAMP phosphodiesterase activity lack other cAMP phosphodiesterase activity. Extracts from such cells thus provide a means by which agents which alter cAMP phosphodiesterases can be identified and isolated.

[0019] It is one of the further objects of this invention to discover, isolate and characterize new genes encoding products that interact with RAS proteins. It is still a further object of this invention to describe methods for identifying agents which inhibit or stimulate the interaction of these new gene products with RAS. This is accomplished through use of methods described herein for cloning genes encoding proteins that interact with RAS, and expressing the encoded proteins in cells that have phenotypes which are sensitive to the activity of these proteins.

[0020] The following is a description of cAMP phosphodiesterases; pathways controlled by RAS proteins; use of the present method for identification of mammalian genes, exemplified by identification of genes encoding cAMP phosphodiesterases and genes encoding products which interact with RAS proteins; use of the method to screen for agents which inhibit or stimulate cAMP phosphodiesterases; screening for agents which inhibit or stimulate interaction of such proteins with RAS proteins; and uses of the invention.

cAMP Phosphodiesterases

[0021] Adenylyl cyclase is an ubiquitous enzyme which generates cyclic adenosine monophosphate (cAMP). cAMP is a universal “second messenger” in both eukaryotes and prokaryotes. In eukaryotes, cAMP exerts its profound effects on cellular physiology by stimulating a cAMP-dependent protein kinase (Robinson, G. A., et al., In Cyclic AMP, Academic Press (1971)). This kinase is composed of regulatory and catalytic subunits. The regulatory subunits combine with and inhibit the catalytic subunits. When the regulatory subunits bind cAMP, they release the catalytic subunits, which in turn phosphorylate proteins on serine and threonine residues. The genes encoding the catalytic and regulatory subunits have been cloned from yeast and mammals (Toda, T., et al., Cell 50: 277 (1987); Shoji, S., et e1., Biochemistry 22: 3702 (1983); Showers, M. O. and Mauver, R. A., J. Biol. Chem. 261: 16288 (1986); Titani, K., et al., Biochemistry 23: 4193 (1984); Takio, K., et al., Biochemistry 23: 4200 (1984)).

[0022] In mammals, cAMP is generated by cells in response to hormones, growth factors, and neurotransmitters (Robison, G. A. et al., Cyclic AMP, Academic Press, New York and London (1971)). The concentration of cAMP in mammalian cells is determined not only by its rate of production by adenylyl cyclase, but also by its rate of degradation by enzymes called phosphodiesterases, or more specifically, cAMP phosphodiesterases.

[0023] A number of important physiological responses in humans are controlled by cAMP levels, including mental function, smooth muscle relaxation, strength of cardiac contractility, release of histamine and other immunoreactive molecules, lymphocyte proliferation and platelet aggregation (Robison, G. A. et al., Cyclic AMP, Academic Press, New York and London (1971)). Thus, the range of diseases which can potentially be affected by agents or pharmaceutical compounds which alter cAMP levels include inflammatory processes (e.g., arthritis and asthma), heart failure, smooth muscle cramps, high blood pressure, blood clotting, thrombosis, and mental disorders. One way to modulate cAMP levels in cells is through the modulation of cAMP phosphodiesterase activity.

[0024] Many drugs which raise cAMP levels in various tissues are in common use. These drugs are useful in treating heart failure, asthma, depression, and thrombosis. Only a few of these drugs appear to work by inhibiting cAMP phosphodiesterases. The pharmaceutical industry has not been notably successful at finding such drugs, since effective drug screens have not been available. The reasons for this are set forth below.

[0025] Most tissues contain so many different isoforms of phosphodiesterases that drug screening based on inhibition of crude tissue extracts is unlikely to yield anything other than a broadly acting inhibitor of phosphodiesterases. Broadly acting inhibitors of cAMP phosphodiesterases, such as theophylline, have many deleterious side effects. A few inhibitors are known which have narrow specificity. Such inhibitors may have great potential utility because they can target phosphodiesterases in one or a few tissue and cell types and thus have a higher therapeutic index.

[0026] The yeast cAMP phosphodiesterase genes PDE1 and PDE2 were the first phosphodiesterase genes cloned (Sass, P., et al., Proc. Natl. Acad. Sci. USA, 83:9303 (1986); Nikawa, J., et al., Mol. Cell. Biol., 7:3629 (1987)). Comparison of the amino acid sequence of the yeast phosphodiesterases to the amino acid sequences of other eukaryotic phosphodiesterases reveals only limited sequence homology. PDE2 has very slight sequence homology to the dunce phosphodiesterase of D. melanogaster and to two phosphodiesterases expressed in bovine heart and brain (Charbonneau, H., et. al., Proc. Natl. Acad. Sci., 83: 9308 (1986)). PDE1 shares even less of this homology, but resembles to a greater extent a secreted form of phosphodiesterase found in D. discoidem (Nikawa, J., et al., Mol. Cell. Biol. 7: 3629 (1987)). Thus, there appear to be many diverse branches of cAMP phosphodiesterase genes in evolution.

[0027] Biochemical, serological and pharmacological studies strongly suggest the existence of multiple families of cAMP phosphodiesterases in mammals (Beavo, J. A. Advances in Second Messenger and Phosphoprotein Research Vol. 22 1 (1988)). Partial amino acid sequence data and recent nucleic acid sequence data confirm this (Charbonneau, H., et al., Proc. Natl. Acad. Sci., USA 83: 9308 (1986); Colicelli, J., et al., Proc. Natl. Acad. Sci. USA 86: 3566 (1989); Davis, R. L., et al., Proc. Natl. Acad. Sci. USA 86: 3604 (1989); Swinnen, J. V., et al., Proc. Natl. Acad. Sci. USA 86: 5325 (1989)).

[0028] The various known phosphodiesterases fall into several classes: (I) Ca⁺⁺/calmodulin dependent, (II) cGMP stimulated, (III) cGMP inhibited, (IV) high affinity cAMP, (V) cGMP and (VI) nonspecific phosphodiesterases. Each class may be made up of a family of related proteins. In some cases these related proteins may be encoded by separate genes and in other cases they may arise from alternative gene splicing. Generally, a tissue expresses multiple classes of phosphodiesterases, which, by their copurification and proteolytic degradation, render biochemical analysis exceedingly difficult. The analysis of this complexity is aided somewhat by the availability of a few pharmacological agents which discriminate between different classes of phosphodiesterases, and, recently, by serological reagents which can distinguish between families and sometimes between members of a family (Beavo, J. A. Advances in Second Messenger and Phosphoprotein Research Vol. 22: 1-38 (1988)).

[0029] The classification of mammalian phosphodiesterases may not be complete, however. New families and types of activities may yet be discovered. The great majority of the cAMP phosphodiesterase genes have not yet been cloned.

Pathways Controlled by RAS Proteins

[0030] The RAS genes were first discovered as the transforming principles of the Harvey and Kirsten murine sarcoma viruses (Ellis, R. W., et al., Nature 292: 506 (1981)). The cellular homologs of the oncogenes of Harvey and Kirsten murine sarcoma viruses (H-RAS and K-RAS) constitute two members of the RAS gene family (Shimizu, K et al., Proc. Natl. Acad. Sci. 80:2112 (1983)). A third member is N-RAS (Shimizu, K. et al., Proc. Natl. Acad. Sci. 80: 2112 (1983)). These genes are known as oncogenes since point mutations in RAS can result in genes capable of transforming non-cancerous cells into cancerous cells (Tabin, C. J., et al., Nature 300: 143 (1982); Reddy, E. P., et al., Nature 300: 149 (1982); Taparowsky, E., et al., Nature 300: 762 (1982);). Many tumor cells contain RAS genes with such mutations (Capon, D. J., et al., Nature 302:33 (1983); Capon, D. J., et al., Nature 304: 507 (1983); Shimizu, K. et al., Nature 304: 497 (1983); Taparowsky, E., et al., Cell 34: 581 (1983); Taparowsky, E., et al., Nature 300: 762 (1982); Barbacid, M., Ann. Rev. Biochem. 56: 779 (1987)).

[0031] Despite the importance of the RAS oncogenes to our understanding of cancer, the function of RAS genes in mammals is not known. The RAS proteins are small proteins (21,000 daltons in mammals) which bind GTP and GDP (Papageorge, A., et al., J. Virol. 44: 509 (1982)). The RAS proteins hydrolyze GTP slowly; specific cellular proteins can accelerate this process (McGrath, J. P., et al., Nature 310: 644 (1984); Trahey, M., et al., Science 238: 542 (1987)). RAS proteins bind to the inner surface of the plasma membrane (Willingham, M. C., et al., Cell 19: 1005 (1980)) and undergo a complex covalent modification at their carboxy termini (Hancock, J. F., et al., Cell 57: 1167 (1989)). The crystal structure of H-RAS is known (De Vos, A. M. et al., Science 239: 888 (1988)).

[0032] The yeast Saccharomyces cerevisiae contains two genes, RAS1 and RAS2, that have structural and functional homology with mammalian RAS oncogenes (Powers, S., et al., Cell 36: 607 (1984); Kataoka, T., et al., Cell 40: 19 (1985) Defeo-Jones, D. et al., Sciene 228: 179 (1985); Dhar, R., et al., Nucl. Acids Res. 12: 3611 (1984)). Both RAS1 and RAS2 have been cloned from yeast plasmid libraries and the complete nucleotide sequence of their coding regions has been determined (Powers, S., et al., Cell 36: 607 (1984); DeFeo-Jones, D., et al., Nature 306: 707 (1983)). The two genes encode proteins with nearly 90% identity to the first 80 amino acid positions of the mammalian RAS proteins, and nearly 50% identity to the next 80 amino acid positions. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% identity for the first 180 positions. After this, at nearly the same position that the mammalian RAS proteins begin to diverge from each other, the two yeast RAS proteins diverge radically. The yeast RAS proteins, like proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid, and X is the terminal amino acid (Barbacid, M., Ann., Rev. Biochem. 56: 779 (1987)). Monoclonal antibody directed against mammalian RAS proteins immumoprecipitates RAS protein in yeast cells (Powers, S., et al., Cell 47: 413 (1986)). Thus, the yeast RAS proteins have the same overall structure and interrelationship as is found in the family of mammalian RAS proteins.

[0033] RAS genes have been detected in a wide variety of eukaryotic species, including Schizosaccharomyces pombe, Dictyostelium discoidem and Drosophila melanogaster (Fukui, Y., and Kaziro, Y., EMBO 4: 687 (1985); Reymond, C. D. et al., Cell 39: 141 (1984); Shilo, B-Z., and Weinberg, R. A., Proc. Natl. Acad. Sci., USA 78: 6789 (1981); Neuman-Silberberg, F., Cell 37: 1027 (1984)). The widespread distribution of RAS genes in evolution indicates that studies of RAS in simple eukaryotic organisms may elucidate the normal cellular functions of RAS in mammals.

[0034] Extensive genetic analyses of the RAS1 and RAS2 of S. cerevisiae have been performed. By constructing in vitro RAS genes disrupted by selectable biochemical markers and introducing these by gene replacement into the RAS chromosomal loci, it has been determined that neither RAS1 nor RAS2 is, by itself, an essential gene. However, doubly RAS deficient (ras1⁻ ras2⁻) spores of doubly heterozygous diploids are incapable of resuming vegetative growth. At least some RAS function is therefore required for viability in S. cerevisiae (Kataoka, T., et al., Cell 37: 437 (1984)). It has also been determined that RAS1 is located on chromosome XV, 7 cM from ADE2 and 63 cM from HIS3; and that RAS2 is located on chromosome XIV, 2 cM from MET4 (Kataoka, T., et al., Cell 37: 437 (1984)).

[0035] Mammalian RAS expressed in yeast can function to correct the phenotypic defects that otherwise would result from the loss of both RAS1 and RAS2 (Kataoka, T., et al., Cell 40: 19 (1985)). Conversely, yeast RAS are capable of functioning in vertebrate cells (De Feo-Jones, D., et al., Science 228: 179 (1985)). Thus, there has been sufficient conservation of structure between yeast and human RAS proteins to allow each to function in heterologous host cells.

[0036] The missense mutant, RAS2^(va119), which encodes valine in place of glycine at the nineteenth amino acid position, has the same sort of mutation that is found in some oncogenic mutants of mammalian RAS genes (Tabin, C. J., et al., Nature 300: 143 (1982); Reddy, E. P., et al., Nature 300: 149 (1982); Taparowsky, E., et al., Nature 300: 762 (1982)). Diploid yeast cells that contain this mutation are incapable of sporulating efficiently, even when they contain wild-type RAS alleles (Kataoka, T., et al., Cell 37: 437 (1984)). When an activated form of the RAS2 gene (e.g., RAS2^(va119)) is present in haploid cells, yeast cells fail to synthesize glycogen, are unable to arrest in G1, die rapidly upon nutrient starvation, and are acutely sensitive to heat shock (Toda, T., et al., Cell 40: 27 (1985); Sass, P., et al., Proc. Natl. Acad. Sci. 83: 9303 (1986)).

[0037]S. cerevisiae strains containing RAS2^(va119) have growth and biochemical properties strikingly similar to yeast carrying the IAC or bcy1⁻ mutations, which activate the cAMP pathway in yeast (Uno, I., et al., J. Biol. Chem. 257: 14110 (1981)). Yeast strains carrying the IAC mutation have elevated levels of adenylate cyclase activity. bcy1− cells lack the regulatory component of the cAMP dependent protein kinase (Uno, I. et al., J. Biol. Chem. 257: 14110 (1982); Toda, T., et al., Mol. Cell. Biol 7: 1371 (1987)). Yeast strains deficient in RAS function exhibit properties similar to adenylate cyclase-deficient yeast (Toda, T., et al., Cell 40: 27 (1985)). The bcy1⁻ mutation suppresses lethality in ras1⁻ ras2⁻ yeast. These results suggest that in the yeast S. cerevisiae, RAS proteins function in the cAMP signalling pathway.

[0038] Adenylyl cyclase has been shown to be controlled by RAS proteins (Toda, T., et al., Cell 40: 27 (1985)). RAS proteins, either from yeast or humans, can stimulate adenylyl cyclase up to fifty fold in in vitro biochemical assays. RAS proteins will stimulate adenylyl cyclase only when bound with GTP (Field, J., et al., Mol. Cell. Biol. 8: 2159 (1988)).

[0039] The phenotypes which are due to activation of RAS, including sensitivity to heat shock and starvation, are primarily the result of overexpression or uncontrolled activation of the cAMP effector pathway via adenylyl cyclase (Kataoka, T., et al., Cell 37: 437 (1984); Kataoka, T. et al., Cell 43: 493 (1985); Toda, T. et al., Cell 40: 27 (1985); Field J., et al., Mol. Cell. Biol., 8: 2159 (1988)). Two S. cerevisiae yeast genes, PDE1 and PDE2, which encode the low and high affinity cAMP phosphodiesterases, respectively, have been isolated (Sass, P., et al., Proc. Natl. Acad. Sci. 83: 9303 (1986); Nikawa, J., et al., Mol. Cell. Biol. 7: 3629 (1987)). These genes were cloned from yeast genomic libraries by their ability to suppress the heat shock sensitivity in yeast cells harboring an activated RAS2^(va119) gene. Cells lacking the PDE genes (i.e., pde1⁻ pde2⁻ yeast) are heat shock sensitive, are deficient in glycogen accumulation, fail to grow on an acetate carbon source, and in general have defects due to activation of the cAMP signaling pathway (Nikawa, J., et al., Mol. Cell. Biol. 7: 3629 (1987)).

[0040] Genetic analysis clearly indicates that RAS proteins have other functions in S. cerevisiae besides stimulating adenylyl cyclase (Toda, T. et al., Japan Sci Soc. Press. Tokyo/VNU Sci. Press, pp. 253 (1987); Wigler, M. et al., Cold Spring Harbor Symposium, Vol. LIII 649 (1988); Michaeli, T., et al., EMBO 8: 3039 (1989)). The precise biochemical nature of these functions is unknown. Experiments with other systems, such as S. pombe and Xenopus laevis oocytes, indicate that RAS stimulation of adenylyl cyclase is not widespread in evolution (Birchmeier, C., et al., Cell 43: 615 (1985)). It is unlikely that RAS stimulates adenylyl cyclase in mammals (Beckner, S. K. et al., Nature 317: 71 (1985)).

Identification of Mammalian-Genes, Exemplified by Genes Encoding cAMP Phosphodiesterases

[0041] The present method can be used to clone a mammalian gene of interest which functions in a microorganism which is genetically altered or defective in a defined manner (an altered microorganism) to correct the genetic alteration or defect and, as a result, modifies an identifiable phenotypic alteration or characteristic associated with the genetic alteration or defect (produces a phenotype more like that of normal or unaltered yeast). Although use of the present method to clone and identify mammalian genes is described in detail in respect to cAMP phosphodiesterases and proteins which interact with RAS proteins, it can be used to clone and identify other mammalian genes which function in an appropriately-selected altered microorganism to correct, complement or supplement the genetic alteration and, as a result, correct the associated phenotypic alteration.

[0042] In its most general form, the method of the present invention is represented in FIG. 1 and can be described as follows: A cDNA library of mammalian mRNAs is produced, using known techniques. This library can be made by cloning double stranded cDNA into an expression vector. The cDNA can be prepared from a preexisting cDNA library, or it can be prepared by the reverse transcription of mRNA purified from a tissue or cell line of choice, using standard procedures (Watson, C. J. and Jackson, J. F. In: DNA cloning, a practical approach, IRL Press Oxford (1984)). This is described in greater detail in Example 1, in which cDNA was derived from rat brain mRNA and in Example 2, in which the cDNA was derived from a human glioblastoma cell line, U1188MG.

[0043] The cDNA obtained is cloned into an expression vector capable of expressing mammalian cDNA inserts as mRNA which can be translated into protein in a host cell of choice. Any expression vector, such as pADNS, into which the cDNA can be inserted and subsequently expressed as mRNA which is translated in an appropriate altered host cell (e.g., altered yeast) can be used. Vectors which have been used for this purpose are described; see FIG. 2A, which is a schematic representation of the expression vector TK161-R2V, whose use is described in Examples 1 and 2, and FIG. 2B, which is a schematic representation of the expression vector pADNS, whose use is described in Example 2. In general, an expression vector contains a transcriptional promoter specific for the host cell into which the vector is introduced For example, the vector used in Examples 1 and 2 contains promoters for expression in S. cerevisiae. The expressed mRNA may utilize the ATG of the cDNA insert as the “start” codon (e.g., the vector of FIG. 2A) or may express the cDNA product as a fusion protein (e.g., the vector of FIG. 2B).

[0044] The cDNA library (present as cDNA inserts in a selected expression vector) is introduced into a host cell of choice, which contains genetic alterations which cause the host cell to have an identifiable phenotypic alteration or abnormality associated with the genetic alteration. The host cell may be a eukaryotic microorganism, such as the yeast S. cerevisiae. Known methods, such as lithium acetate-induced transformation, are used to introduce the cDNA-containing expression vector. The genetic alterations may lead to defects in the metabolic pathways controlled by the RAS proteins and the associated readily discernible phenotype may be sensitivity to heat shock or nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on certain carbon sources, failure to sporulate, failure to mate, or other properties associated with defects in the pathways controlled by RAS proteins. For example, as described in Examples 1 and 2, the genetic alteration can be the presence of the RAS2^(va119) gene. Yeast containing such an alteration exhibit heat shock sensitivity, which, as described in Examples 1 and 2, can be overcome by expression of mammalian genes. Other genetic alterations can be chosen, such as disruptions of the PDE1 and PDE2 genes in S. cerevisiae or disruptions of, or the presence of an activated allele of, ras1 in S. pombe. Different genetic alterations in the host cell may be correctable by different subsets of mammalian cDNA genes.

[0045] After introduction of the cDNA insert-containing expression vector, host cells are maintained under conditions appropriate for host cell growth. Those host cells which have been corrected for their phenotypic alteration are selected and the mammalian gene which they express is recovered (e.g., by transformation of E. coli with DNA isolated from the host cell). The mammalian gene is isolated and can be sequenced and used for further analysis in a variety of ways. For example, the encoded protein can be identified and expressed in cultured cells for use in further processes.

[0046] The present method has been used, as described in Examples 1 and 2, to isolate new mammalian genes whose presence in yeast cells has resulted in correction of a phenotypic alteration associated with a genetic alteration (the presence of the RAS2^(va119) gene). The nucleotide sequences of these genes, as well as the amino acid sequence encoded by each, are described in Examples 1 and 2, and are shown in FIGS. 3-7. The genes of FIGS. 3 and 4 are homologous to the D. melanogaster dunce gene. The gene of FIG. 3 has also been recently isolated by others using the D. melanogaster dunce gene as probe (Swinnen, J. V., et al., Proc. Natl. Acad. Sci. 86: 5325 (1989)).

Screening and Identification of Agents which Alter cAMP Phosphodiesterase Activity

[0047] In its most general form, the second part of the invention (pharmacological screening) is carried out as follows: It is possible to screen for agents that reduce or stimulate the activity of any mammalian protein whose presence or expression in an altered microbial host cell in which a genetic alteration is associated with an identifiable phenotypic alteration results in correction of the phenotypic alteration. Two types of screens are possible, and are illustrated in Examples 3 and 4.

[0048] The first type of pharmacological screen is applicable when the mammalian gene encodes a protein of known and assayable biochemical function. The mammalian gene is first expressed in a microbial host by utilizing an appropriate host expression vector of the type already described. Extracts of host cells are prepared, using known techniques; the cells are disrupted and their cellular constituents released. Crude cellular extract or purified mammalian protein is assayed for the known biochemical function in the presence of agents, the effects of which on the protein are to be assessed. In this manner, agents which inhibit or stimulate the activity of the mammalian protein can be identified.

[0049] This type of procedure can be carried out to analyze the effects of selected agents on mammalian cAMP phosphodiesterases. For example, a yeast strain lacking both endogenous PDE1 and PDE2 genes can be used as the host cell, into which cDNA encoding mammalian cAMP phosphodiesterase is introduced in an appropriate expression vector and expressed. Such a host cell is particularly useful because there is no background cAMP phosphodiesterase activity (Colicelli, J., et al., Proc. Natl. Acad. Sci. USA 86:3599 (1989)) and hence activity of the mammalian enzyme can be cleanly assayed even in crude cell extracts. This procedure is illustrated in Example 3, in which it is demonstrated that the enzymatic activity of the rat DPD gene product is inhibited by the pharmacological agents Rolipram and R020 1724, but not by the pharmacological agent theophylline.

[0050] The second type of pharmacological screen is applicable even when the mammalian gene encodes a protein of unknown function, and, thus, cannot be assayed by a biochemical activity. In this method, agents to be tested are applied or introduced directly to the genetically altered microbial host expressing the mammalian protein. Agents capable of inhibiting the mammalian gene or gene product are identified by their ability to reverse the phenotype originally corrected by expression of the mammalian protein in the altered host.

[0051] This procedure has been used for mammalian cDNAs encoding cAMP phosphodiesterases and a yeast containing RAS2^(va119) as the host strain (see Example 4). This host is heat shock sensitive. When the rat DPD gene is introduced into the heat shock sensitive host and expressed, the host strain becomes resistant to heat shock. When the now—resistant cells are incubated in Rolipram, they become heat shock sensitive again, indicating that Rolipram inhibits the activity of the rat DPD gene product. This pharmacological screen does not require that the function of the DPD gene product be known. This same approach can be applied to the genes which are the products of Example 2.

Applications of the Present Method and Products

[0052] The present method is useful for cloning novel mammalian genes which encode cAMP phosphodiesterases or proteins which interact with a RAS protein. As described, novel mammalian genes have been cloned, using the present method, and the amino acid sequence of the encoded protein has been deduced. Other mammalian genes encoding additional cAMP phosphodiesterases or additional proteins which interact with a RAS protein can be cloned using the method described. All or a portion of the sequence of the mammlaian genes encoding cAMP phosphodiesterases can be used as probes, in known techniques, to identify homologs and the products encoded by such assayed homologs as described herein for cAMP phosphodiesterase activity. Similarly, all or a portion of the mammalian genes encoding products which interact with RAS proteins can be used to identify homologs and the ability of the encoded proteins to interact with RAS proteins assessed as described herein.

[0053] Alternatively, mammalian genes encoding other proteins which function in an altered microorganism to correct, complement or supplement the altered or defective genetic activity can be cloned, using a microorganism with an appropriately-selected alteration (e.g., a change in a different biochemical pathway) which is associated with an identifiable phenotypic characteristic.

[0054] The present invention is also useful for identifying agents, particularly chemical compounds, which alter (reduce or stimulate) cAMP phosphodiesterase and, thus, affect cAMP activity (e.g., by causing more rapid cAMP breakdown or inhibiting cAMP breakdown and, thus, shortening or prolonging the duration of cAMP activity, respectively). The present method is also useful for identifying agents which alter (inhibit or enhance) the interaction of gene products with RAS proteins.

[0055] Antibodies specific for proteins encoded by the mammalian genes isolated using the present method can be produced, using known techniques. Such antibodies may be polyclonal or monoclonal and can be used to identify cAMP phosphodiesterases or proteins which interact with RAS proteins (e.g., the same proteins as those encoded by the mammalian genes or proteins sufficiently similar to the encoded proteins that they are recognized or bound by an antibody raised against the encoded proteins).

[0056] The present invention will now be illustrated by the following examples, which are not intended to be limiting in any way.

EXAMPLE 1 Identification of a Mammalian Gene that Can Revert the Heat Shock Sensitivity of RAS2^(va119) Yeast

[0057] Several yeast genes have been isolated which, when overexpressed on extrachromosomal yeast vectors, are capable of suppressing the heat shock sensitivity exhibited by the RAS2^(va119) expressing strain TK161-R2V (Sass, P., et al., Proc. Natl. Acad. Sci. USA 83 (1986); Nikawa, J., et al., Mol. Cell. Biol., 7:3629 (1987)). As described in this example, mammalian genes that can function in yeast to render RAS2^(va119) cells resistant to heat shock have now been isolated. A rat brain cDNA library was produced and cloned into the yeast expression vector, pADNS (FIG. 1A). Double stranded cDNAs were prepared and ligated to NotI linkers, cleaved with NotI restriction enzyme, and cloned into pADNS at the NotI site situated between the alcohol dehydrogenase promoter and termination sequences of the vector. The use of the rare cutting NotI obviated the need for restriction site methylases commonly used in cDNA cloning.

[0058] Approximately 1.5×10⁵ independent cDNA inserts were contained in the library, with an average insert size of 1.5 kbp. DNA prepared from the cDNA expression library was used to transform the RAS2^(va119) yeast strain, TK161-R2V. 50,000 Leu⁺ transformants obtained were subsequently tested for heat shock sensitivity. Only one transformant displayed heat shock resistance which was conditional upon retention of the expression plasmid. A plasmid, pADPD, was isolated from this transformant and the 2.17 kb NotI insert was analyzed by restriction site mapping and nucleotide sequencing (FIG. 2).

[0059] A large open reading frame of 562 codons was found. The first ATG appears at codon 46 and a protein which initiates at this codon would have a predicted molecular weight of approximately 60 kDa. This gene is designated DPD. A search for similar sequences was performed by computer analysis of sequence data banks, and the Drosophila melanogaster dunce gene was found. The two genes would encode proteins with an 80% amino acid identity, without the introduction of gaps, over a 252 amino acid region located in the center of the rat DPD cDNA. The dunce gene has been shown to encode a high affinity cAMP phosphodiesterase (Chen, C., et al., Proc. Natl. Acad. Sci. USA 83:9313 (1986); Davis, R. L. and Kiger, J. A. J. Cell Biol. 90:101 (1981); Walter, M. F. and Kiger, J. A. J. Neurosci. 4:494 (1984)).

[0060] In order to demonstrate that the sequences upstream and downstream of the large sequence identity region were in fact contiguous with that region in the mRNA, rather than artifacts of the method for cDNA cloning, the structure of the cloned cDNA was compared with the structure of DPD cDNAs contained in an independently prepared, first strand cDNA population obtained by reverse transcribing total rat brain poly (A)⁺ RNA with an oligo dT primer. Oligonucleotide primers complementary to sequences located within the identity region, and to sequences near the 5′ or 3′ ends of the coding strand, were made. Using either the cloned DPD DNA or the total first strand cDNA material as template, polymerase chain reactions (PCR) were carried out using four different primer sets and the reaction products were analysed by polyacrylamide gel electrophoresis. In each case, a fragment of the predicted length was obtained using either of the template DNAs. The band assignments were confirmed by cleavage with restriction endonucleases having recognition sites within the amplified DNA product. Again, in each case, the primary PCR product obtained using either source of template yielded cleavage products of the predicted sizes. The results indicate that the sequence arrangement in the cloned cDNA faith-fully reflects the structure of the rat mRNA.

Expression and Characterization of the DPD Gene Product

[0061]S. cerevisiae encodes two cAMP phosphodiesterase genes, PDE1 and PDE2 (Sass, P., et al., Proc. Natl. Acad. Sci. USA 83:9303 (1986); Nikawa, J., et al., Mol. Cell. Biol. 7:3629 (1987)). The S. cerevisiae strain 10 DAB carries disruptions of both of these genes. The resulting cAMP phosphodiesterase deficiency leads to elevated intracellular cAMP levels and a heat shock sensitivity phenotype similar to that of strains harboring the RAS2^(va119) allele (Nikawa, J., et al., Mol. Cell. Biol. 7:3629 (1987). 10 DAB cells were transformed with the DPD expression plasmid, pADPD, and assayed for heat shock sensitivity. Expression of the rat DPD gene indeed rendered this host resistant to heat shock.

[0062] In order to analyse the biochemical properties of the DPD gene product, crude cell extracts were prepared from one liter cultures of 10 DAB which had been transformed with either pADNS or pADPD. Phosphodiesterase activity assays were performed using cAMP as substrate. Control extracts (10 DAB with pADNS) showed no cAMP phosphodiesterase activity. Results with the controls were unchanged when performed at 0° C. or in the absence of Mg²⁺ and were comparable to results obtained when no extract was added. These results indicate that there is no detectable background phosphodiesterase activity in strain 10 DAB.

[0063] In contrast, considerable cAMP phosphodiesterase activity was seen in the 10 DAB strain transformed with pADPD. The rate of cAMP hydrolysis in cells containing DPD was measured as a function of cAMP concentration. The deduced Km for cAMP is 3.5 μM and the calculated Vmax is 1.1 nmol/mg/min.

[0064] The assay conditions were varied in order to ascertain the cation preferences of the enzyme and to determine the ability of calcium and calmodulin to stimulate its activity. In these assays, Mn²⁺ can be utilized as well as Mg²⁺, and either cation in 1 mM final concentration was sufficient. Calcium/calmodulin was unable to stimulate the measured phosphodiesterase activity in the extract. A parallel assay using beef heart phosphodiesterase (Boeringer Mannheim) yielded a 6.5 fold stimulation with the addition of calcium/calmodulin. Finally, no cGMP phosphodiesterase activity was detected in these assays. Beef heart phosphodiesterase was again used as a positive control. In addition, cGMP present in amounts 100 fold over substrate concentrations was unable to inhibit cAMP phosphodiesterase activity.

Strains, Media, Transformations and Heat Shock

[0065]Escherichia coli strain HB101 was used for plasmid propagation and isolation, and strain SCS1 (Stratagene) was used for transformation and maintenance of the cDNA library (Mandel, M., and Higa, A. J. Mol. Biol. 53: 159 (1970); Hanahan, D. J. Mol. Biol. 166:557 (1983)). Saccharomyces cerevisiae strain TK161-R2V (MAT a leu2 his3 ura3 trp1 ade8 can1 RAS2^(va119) ) (Toda, T., et al., Cell 40:27 (1985) and strain 10 DAB were used. Strain 10 DAB was created from a segregant of a diploid strain produced by mating TS-1 (Kataoka, T., et al., Cell 40:19 (1985)) and DJ23-3C (Nikawa, J. I., et al., Genes and Development 1:931 (1987)). The segregant (MATα leu2 his3 ura3 ade8 pde1::LEU2 pde2::URA3 ras1::HIS3) was subsequently transformed with the 5.4 kbp Xba1 pde1::ADE8 fragment of pYT19DAB to yield strain 10DAB. Yeast cells were grown in either rich medium (YPD) or synthetic medium with appropriate auxotrophic supplements (SC) (Mortimer, R. K. and Hawthorne, D. C. In: The Yeast, vol. 1 385 (1969)). Transformation of yeast cells was performed with lithium acetate (Ito, H., et al., J. Bacteriol., 153:163 (1983)). Heat shock experiments were performed by replica plating onto preheated SC plates which were maintained at 55° C. for 10 minutes, allowed to cool, and incubated at 30° C. for 24-48 hrs. Segregation analysis was performed by growing yeast transformants in YPD for 2-3 days, plating onto YPD plates, and replica plating onto YPD, SC-leucine (plasmid selection), and YPD heat shock plates.

Plasmids, DNA Manipulations and Sequencing

[0066] Plasmid DNA from individual E.coli colonies was purified by standard procedures (Holmes, D. S., and Quigley, M. Anal. Biochem 114 193 (1981); Katz, L., et al., J. Bacteriol. 114 477 (1973). Extrachromosomal DNA was isolated from yeast as previously described (Nikawa, J. et al., Mol. Cell. Biol., 7:3629 (1987)). The plasmid pYT19DAB was constructed from pYT19 (Nikawa, J. et al., Mol. Cell. Biol., 7:3629 (1987)) by first deleting PDE1 sequences between the SmaI and BalI restriction sites to yield pYT19D. The 4 kbp BamHI fragment of the ADE8 gene was then inserted into the BamHI site of pYT19D to yield pTY19DAB. The cloning vector pADNS is based on the plasmid pAD1 previously described (Powers, S., et al., Cell 47:413 (1986)). pADNS consists of a 2.2 kbp Bgl II to Hpal fragment containing the S. cerevisiae LEU2 gene from YEp213 (Sherman, F., Fink, et al., Laboratory Course Manual for Methods in Yeast Genetics, eds. Sherman, F., Fink, G. R. and Hicks, J. B., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1986)), a 1.6 kbp HpaI to HindIII fragment of the S. cerevisiae 2μ plasmid containing the origin of replication, and a 2.1 kbp SspI to EcoRI fragment containing the ampicillin resistance gene from the plasmid pUC18. It also contains a 1.5 kbp BamHI to HindIII fragment of the modified S. cerevisiae alcohol dehydrogenase (ADH1J) promoter (Bennetzen, J. L. and Hall. B. D. J. Biol. Chem. 257:3018 (1982); Ammerer, G. Meth. Enzymol. 101:192 (1983)) and a 0.6 kbp HindIII to BamHI fragment containing the ADH1 terminator sequences. The promoter and terminator sequences are separated by a polylinker that contains the restriction endonuclease sites NotI, SacII, and SfiI between the existing HindIII and SacI sites. The oligonucleotides used to create these sites were 5′-GGCCAAAAAGGCCGCGGCCGCA and 5′-TCGACCGGTTTTTCCGGCGCCGGCGTTCGA. The plasmid pADPD is a pADNS-derived plasmid containing the 2.17 kbp DPD cDNA insert.

[0067] Sequencing was performed using the dideoxy chain termination method (Sanger, R., et al., Proc. Natl. Acad. Sci. USA 74:5463 (1977); Biggin, M. D., et al., Proc. Natl. Acad. Sci. USA 80:3963 (1983)). Genalign was used to align the DPD and dunce sequences (GENALIGN is a copyrighted software product of IntelliGenetics, Inc.; developed by Dr. Hugo Martinez). RNA was purified from Sprague-Dawley rat brains by published procedures (Chirgwin, J. M. et al., Biochem. 18:5294 (1979); Lizardi, P. M. Methods Enzymol 96:24 (1983); Watson C. J. and Jackson J. F. In: DNA cloning, a practical approach, IRL Press Oxford (1984)). cDNAs were ligated to the NotI linker oligonucleotides 5′-AAGCGGCCGC and 5′-GCGGCCGCTT. The cDNAs were cleaved with NotI and cloned into the NotI site of pADNS using standard procedures.

[0068] Polymerase chain reactions (PCRs) were carried out in thermocycler (Perkin Elmer, Cetus) using a modification of published procedures (Saiki, R., et al., Science 239:487 (1988)). Reaction mixtures contained template DNA (1 ng of cloned DNA, or 1 pg of total first strand cDNA), 25 pmoles of oligonucleotide primers, 200 μM deoxyribonucleotide triphosphates, 10 mM Tris HCl (Ph8.4), 50 mM KCl, 3 mM MgCl₂, and 0.01% (w/v) gelatin. The oligonucleotide primers used, as designated in FIG. 4, were:

[0069] A, 5′-CACCCTGCTGACAAACCT⁴⁴;

[0070] B, 5′-ATGGAGACGCTGGAGGAA¹⁵³;

[0071] C, 5′-ATACGCCACATCAGAATG⁶⁷⁶;

[0072] D, 5′-TACCAGAGTATGATTCCC¹⁴⁴⁹;

[0073] E, 5′-GTGTCGATCAGAGACTTG¹⁶⁶⁸ and

[0074] F, 5′-GCACACAGGTTGGCAGAC²⁰⁴⁸. The numbers indicate position coordinates in FIG. 3. Primers C, E and F are non-coding strand sequences. Thirty cycles (1.5 min at 94° C., 3 min at 55° C., and 7 min at 72° C.) were performed and the reaction products were analysed by polyacrylamide gel electrophoresis.

Phosphodiesterase Assays

[0075] Yeast cells were grown at 30° C. for 36 hours in one liter cultures of synthetic media (SC-leucine). Cells were harvested and washed with buffer C (200 mM MES, 0.1 mM MgCl₂, 0.1 mM EGTA, 1 mM β-mercaptoethanol), were resuspended in 30 ml buffer C with 50 μl 1M PMSF, and were disrupted with a French press. The extracts were centrifuged at 1,600 g for 10 min and the supernatants were spun at 18,000 g for 90 min (4° C.). The supernatant was assayed for phosphodiesterase activity (Sass, P., et al., Proc. Natl. Acad. Sci. USA 83:9303 (1986); Nikawa, J., et al., Mol. Cell. Biol. 7:3629 (1987)). All the reactions contained Tris-HCl (pH7.5) (100 mM), cell extract (50 μg protein/ml), 5′-nucleotidase (Sigma, 20 ng/ml) and 10 MM Mg²⁺ (unless otherwise stated) and the indicated cyclic nucleotide concentrations. Assays for the cGMP hydrolysis used 1.5 μM cGMP. Inhibition studies employed 5 μM cAMP in the presence of varying amounts of cGMP up to 500 μM. [³H]cAMP and [³H]cGMP were obtained from NEN (New England Nuclear). Reactions were incubated for 10 min at 30° C. and stopped with 5× stop solution (250 mM EDTA, 25 mM AMP, 100 mMcAMP).

Discussion

[0076] Previous workers have cloned a mammalian gene in yeast using a biological screen (Lee, M. G. and Nurse, P. Nature 327:31 (1987)). In that case, a homolog to the cdc2 gene of S. pombe was cloned by screening a cDNA library for complementation of cdc2 mutants. In that library, the cDNAs were inserted proximal to the SV40 early T antigen promoter. In our work we have employed a library with mammalian cDNAs inserted into a yeast expression vector, proximal to a strong yeast promoter. In addition, we have employed NotI linkers for cDNA cloning, which allows the convenient subcloning of an entire insert library from one vector to another. We feel that this will be a generally useful approach for cloning genes from higher eukaryotes when functional screens are possible in yeast. This system is particularly useful for the cloning of other cAMP phosphodiesterases from mammals. The availability of yeast strains totally lacking endogenous cAMP phosphodiesterase activity will also facilitate the biochemical characterization of these new phosphodiesterases.

[0077] The mammalian DPD cDNA can encode a protein with a high degree of amino acid sequence identity (80%) with the predicted D. melanogaster dunce gene product over an extended region. The dunce gene has been shown to encode a high affinity cAMP phosphodiesterase required for normal learning and memory in flies (Chen, C., et al., Proc. Natl. Acad. Sci. USA 83:9313 (1986); Davis R. L. and Kiger, J. A. J. Cell Biol. 90:101 (1981); Walter, M. F. and Kiger, J. A. J. Neurosci. 4:495 (1984)). Compared to the striking level of sequence identity between DPD and dunce, the sequence conservation among other known cAMP phosphodiesterases is scant (Charbonneau, H., et al., Proc. Natl. Acad. Sci. USA 83:9308 (1986)). Therefore the DPD-dunce homology in the conserved region represents more than a constraint on sequences required for cAMP binding and hydrolysis, and suggests a conservation of interactions with other components.

[0078] Biochemical characterization of the DPD cDNA product expressed in yeast indicates that it is a high affinity cAMP specific phosphodiesterase, as is dunce (Davis, R. L. and Kiger, J. A. J. Cell. Biol. 90:101 (1981); Walter M. F. and Kiger, J. A. J. Neurosci. 4 (1984)). In addition, DPD activity, as measured in our assays, is not stimulated by the presence of calcium/calmodulin. This property is shared with dunce and is distinct from some other phosphodiesterases (Beavo, J. A. In Advances in second messenger and phosphorprotein research, eds. Greengard, P. and Robinson, G. A., Raven Press, NY vol. 22 (1988)). The two proteins, DPD and dunce, thus appear to have similar biochemical characteristics. However, it should also be noted that DPD encodes a protein product which shows much less significant homology (35%) to dunce beyond the previously described highly conserved core region. These non-conserved sequences could result in an altered or refined function for this mammalian dunce homolog.

[0079] The DPD sequence encodes a methionine codon at position 46 and the established reading frame remains open through to position 563, resulting in a protein with a predicted molecular weight of 60 kDa. The same reading frame, however, is open beyond the 5′ end of the coding strand (FIG. 2). At present, it is not known if the methionine codon at position 46 is the initiating condon for the DPD protein. The coding sequence is interrupted by three closely spaced terminator codons. However, the established reading frame then remains open for an additional 116 codons, followed by more terminator codons, a polyadenylation consensus signal and a polyadenine stretch. This 3′ open reading frame could be incorporated into another dunce-like phosphodiesterase through alternate splicing.

[0080] Davis et al., (Davis, R. L. et al., Proc. Natl. Acad. Sci. USA 86:3604 (1989)) have also isolated a mammalian dunce homolog from a rat brain cDNA library using standard nucleic acid hybridization techniques. The gene which they describe is indeed similar to, through distinct from, the DPD cDNA described here. Within the highly conserved region, as defined in this work, the predicted amino acid sequences of the two rat genes are 93% identical. This homology falls off dramatically, however, in the flanking regions which show amino acid identities of 60% (upstream) and 30% (downstream) and require the use of sequence gaps for optimum alignment.

EXAMPLE 2 Identification of a Human Gene that Can Revert the Heat Shock Sensitivity of RAS2^(va119) Yeast

[0081] A cDNA library was constructed in λZAP using NotI linkers. In this example, the cDNA derived from mRNA purified from the human glioblastoma cell line U118MG. Inserts from the λ vector were transferred into two yeast expression vectors. One, pADNS, is as described before. The other, pADANS (see FIG. 2B), differs in that the mRNA expressed will direct the synthesis of a fusion protein: an N terminal portion derived from the alcohol dehydrogenase protein and the remainder from the mammalian cDNA insert. Thus, two mammalian cDNA expression libraries were constructed.

[0082] These libraries were screened, as in the previous example, for cDNAs capable of correcting the heat shock sensitivity of the S. cerevisiae host TK161-R2v. Several cDNAs were isolated and analysed by sequencing. Four different cDNA genes were thereby discovered, and their sequences are shown in FIGS. 4-7.

[0083] The gene of FIG. 4 (JC44) was shown by computer analysis to be homologous to the rat DPD gene. Biochemical analysis has proven that JC44 encodes a cAMP phosphodiesterase. The other genes, called JC99, JC265, and JC310, show no significant homology to previously isolated genes.

[0084] The genes of FIGS. 3 and 4 were shown to be able to correct the phenotypic defects of pde1⁻ pde2⁻ yeast strains. The genes of FIGS. 5-7 were unable to do so. Thus, it appears that the latter genes do not encode cAMP phosphodesterases. Rather, these genes encode proteins of unknown function which appear to be able to correct phenotypic defects in yeast with activated RAS proteins.

Materials and Methods

[0085] Procedures of Example 1 were followed throughout. Described here is the construction of the plasmid pADANS, shown in FIG. 2B. A PCR reaction was carried out on the yeast ADH1 gene in pJD14 (Bennetzen, J. L. and Hall, B. D. J. Biol. Chem. 257:2018 (1982)). One oligonucleotide primer (5′ TCTAAACCGTGGAATATT) was placed within the promoter region of the gene. The second primer (5′ GTCAAAGCTTCGTAGAAGATAACACC) was designed to hybridize within the coding region of the gene. This primer included 5′ non-hybridizing sequence encoding a HindIII endonuclease recognition site. The PCR product was then purified, digested with HindIII and EcoRV and ligated into the 8.0 kb HindIII and EcoRV (partial) digested fragment of pADNS. The resulting plasmid, pADANS, contains the entire ADH1 promoter and the first 14 amino acid codons of the ADH1 gene followed by the HindIII and NotI restriction endonuclease sites.

EXAMPLE 3 Identification of Agents which Inhibit Phosphodiesterase Activity

[0086] This example illustrates the use of the genes and cells described in Example 1 to identify chemical compounds which inhibit the activity of a known enzyme, the rat DPD phosphodiesterase. To test the efficiency of known inhibitory compounds, cell free extracts were made as described in Phosphodiesterase Assays. Yeast cells deficient in endogenous phosphodiesterase (10 DAB), and expressing the rat DPD or yeast PDE2 genes from the described expression vector, were used. One liter cultures were harvested, washed in buffer C (20 mM MES/0.1 mM MgCl₂/0.1 mM EGTA/1 mM 2-mercaptoethanol), resuspended in buffer C containing 1.5 mM phenylmethylsulfonyl fluoride, and disrupted in a French press at 4° C. Cell extracts were clarified at 100 g for 10 minutes and at 18000 g for 90 minutes. PDE activities were assayed as published (Saiki et al., Science 239:487-491 (1988); Charbonneau et al., Proc. Natl. Acad. Sci. USA 83:9308-9312 (1986); Tempel et al., Proc. Natl. Acad. Sci. USA 80:1482-1486 (1983)) in a reaction mix containing 50 μg of cell protein/ml, 100 mM Tris (pH 7.5), 10 mM Mg⁺⁺, 5 μM 5′-nucleotidase and [³]cAMP Hydrolysed AMP was separated from cAMP using AG1-X8 resin from Bio Rad. About 10⁴ cpm were obtained for 10 minutes reactions and backgrounds (phosphodiesterase deficient-yeast or no extract) were about 300 cpm. The cytosolic fraction was assayed in the presence or absence of inhibitory compounds. These assays measure the amount of adenosine 5′ monophosphate (AMP) produced by phosphodiesterase-catalysed hydrolysis of adenosine 3′, 5′-cyclic adenosine monophosphate (cAMP). For each extract the percent inhibition for various concentrations of known inhibitors is given in Table 1. The percent inhibition represents the decrease in phosphodiesterase activity relative to measurements made in the absence of inhibitors. Rolipram, and the related compound R020 1724, were the most effective inhibitors of DPD activity. TABLE 1 Inhibition of Phosphodiesterases by Chemicals Phosphodiesterase Agent Concentration (μM) Inhibition (%) PDE2 Theophylline 250 0.0 IBMX 250 0.0 R020 1724 100 3.0 Rolipram 100 0.0 DPD-1 Theophylline 250 42. IBMX 250 87. R020 1724 0.1 35. 1.0 52. 10.0 79. 100.0 92. Rolipram 0.1 50. 1.0 72. 10.0 92. 100.0 95.

[0087] This analysis can, of course, be extended to test new or related chemical compounds for their ability to inhibit DPD activity, or the activity of another phosphodiesterase expressed in this system. Clearly, this form of analysis can also be extended to other genes cloned and expressed in a similar manner, for which there is an assayable enzymatic activity.

EXAMPLE 4 Identification of Agents which Inhibit Mammalian Proteins of Unknown Function Expressed in Yeast

[0088] This example illustrates the use of the genes and methods described to identify chemical compounds which inhibit the function of the encoded mammalian proteins expressed in yeast, even when the function of that protein is not known. 10DAB cells, which are phosphodiesterase deficient, are sensitive to heat shock. As already discussed, when these cells express DPD, they become resistant to heat shock. FIG. 8 demonstrates the inhibition of DPD function in yeast cells assayed by heat shock survival. 10 DAB cells expressing DPD were maintained in rich medium (YPD) for three days at stationary phase. These cultures were then treated with rolipram, a known phosphodiesterase inhibitor, for 40 minutes at a final concentration of 100 μM. Control cultures were not treated with any inhibitor. These cultures were then heat shocked in glass tubes at 50° C. for 30 minutes. One microliter of each culture was plated. As shown in FIG. 8, cultures treated with rolipram (right side) were much more sensitive to heat shock, reflecting an inhibition of DPD enzymatic function.

[0089] Similarly, the suppression of heat shock sensitivity in the RAS2^(va119) yeast strain (TK161-R2V) by DPD expression will also be inhibited by drug treatment. In addition, any other phenotype which is dependent on DPD phosphodiesterase activity should be affected by the presence of the inhibitory drug. The effect of a drug or agent can be assessed as described. Finally, in the most generalized case, inhibitory chemicals for proteins of unknown function, expressed from mammalian cDNAs in yeast, can be discovered in a similar way. This approach depends only on the phenotype consequent to expression of the protein and not on knowledge of its function.

Equivalents

[0090] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

1 20 1 2156 DNA Rattus norvegicus CDS (134)..(1684) 1 cttgcgaatc gtaagaaaca atttcaccct gctgacaaac cttcacggag caccgaacaa 60 gaggtcgcca gcggctagtc aggctccagt caccagagtc agcctgcaag aagaatcata 120 tcagaaacta gca atg gag acg ctg gag gaa cta gac tgg tgc cta gac 169 Met Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp 1 5 10 cag cta gag acc atc cag acc tac cgc tct gtc agc gag atg gct tca 217 Gln Leu Glu Thr Ile Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser 15 20 25 aac aag ttc aaa agg atg ctg aac cgg gag ctg aca cac ctc tca gag 265 Asn Lys Phe Lys Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu 30 35 40 atg agc aga tca ggg aac caa gtg tct gaa tac att tcg aac acg ttc 313 Met Ser Arg Ser Gly Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe 45 50 55 60 tta gac aag cag aac gat gtg gaa atc cca tct ccc acc cag aag gac 361 Leu Asp Lys Gln Asn Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp 65 70 75 agg gag aag aag aag aag cag cag ctc atg acc cag ata agt gga gtg 409 Arg Glu Lys Lys Lys Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val 80 85 90 aag aaa ctg atg cac agc tca agc ctg aac aac aca agc atc tca cgc 457 Lys Lys Leu Met His Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg 95 100 105 ttt gga gtc aac acg gaa aat gag gat cat cta gcc aag gag ctg gaa 505 Phe Gly Val Asn Thr Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu 110 115 120 gac ctg aac aaa tgg ggc ctt aac atc ttc aac gtg gct ggg tac tcc 553 Asp Leu Asn Lys Trp Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser 125 130 135 140 cat aat cgg ccc ctc aca tgc atc atg tac gcc att ttc cag gaa aga 601 His Asn Arg Pro Leu Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg 145 150 155 gac ctt cta aag acg ttt aaa atc tcc tcc gac acc ttc gta acc tac 649 Asp Leu Leu Lys Thr Phe Lys Ile Ser Ser Asp Thr Phe Val Thr Tyr 160 165 170 atg atg act tta gaa gac cat tac cat tct gat gtg gcg tat cac aac 697 Met Met Thr Leu Glu Asp His Tyr His Ser Asp Val Ala Tyr His Asn 175 180 185 agc ctg cac gct gct gac gtg gcc cag tca acg cac gtt ctc ctc tct 745 Ser Leu His Ala Ala Asp Val Ala Gln Ser Thr His Val Leu Leu Ser 190 195 200 acg cca gca ctg gat gct gtc ttc aca gac ctg gaa atc ctg gct gcc 793 Thr Pro Ala Leu Asp Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala 205 210 215 220 att ttt gca gct gcc atc cat gat gtt gat cat cct gga gtc tcc aat 841 Ile Phe Ala Ala Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn 225 230 235 cag ttt ctc atc aat aca aat tcc gaa ctt gct ttg atg tat aat gac 889 Gln Phe Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp 240 245 250 gaa tct gtg ctg gaa aac cat cac ctc gct gtg gga ttc aag ctc ctt 937 Glu Ser Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu 255 260 265 caa gag gaa cat tgc gac atc ttt cag aat ctt acc aag aag caa cgc 985 Gln Glu Glu His Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg 270 275 280 cag aca ctc agg aaa atg gtg att gac atg gtg tta gca act gat atg 1033 Gln Thr Leu Arg Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met 285 290 295 300 tcc aag cac atg agc ctc ctg gct gac ctt aaa acg atg gta gaa acc 1081 Ser Lys His Met Ser Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr 305 310 315 aaa aag gtg acg agc tcc ggt gtt ctc ctc ctg gac aac tat act gac 1129 Lys Lys Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp 320 325 330 cgg ata cag gtt ctt cgc aac atg gta cat tgt gca gac ctg agc aac 1177 Arg Ile Gln Val Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn 335 340 345 cct acc aag tcc ttg gag ttg tat cgg caa tgg act gat cgc atc atg 1225 Pro Thr Lys Ser Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met 350 355 360 gag gag ttt ttc caa cag gga gac aaa gaa cgg gag agg gga atg gag 1273 Glu Glu Phe Phe Gln Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu 365 370 375 380 att agc cca atg tgt gat aaa cac aca gct tct gtg gaa aag tcc cag 1321 Ile Ser Pro Met Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln 385 390 395 gtt ggt ttc att gac tac att gtc cat cca ttg tgg gag acc tgg gca 1369 Val Gly Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala 400 405 410 gac ctg gtt cag cct gat gct caa gac att ttg gac aca cta gaa gat 1417 Asp Leu Val Gln Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp 415 420 425 aac agg aac tgg tac cag agt atg att ccc cag agc ccc tct cca cca 1465 Asn Arg Asn Trp Tyr Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro 430 435 440 ctg gac gag agg agc agg gac tgc caa ggc ctt atg gag aag ttt cag 1513 Leu Asp Glu Arg Ser Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln 445 450 455 460 ttc gaa ctg acc ctt gaa gaa gag gat tct gaa gga ccg gaa aag gag 1561 Phe Glu Leu Thr Leu Glu Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu 465 470 475 gga gaa ggc ccc aac tat ttc agc agc aca aag aca ctt tgt gtg atc 1609 Gly Glu Gly Pro Asn Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile 480 485 490 gat cca gag aac agg gat tct ctg gaa gag act gac ata gac att gcc 1657 Asp Pro Glu Asn Arg Asp Ser Leu Glu Glu Thr Asp Ile Asp Ile Ala 495 500 505 aca gaa gac aag tct ctg atc gac aca taatctccct ctgtgtggag 1704 Thr Glu Asp Lys Ser Leu Ile Asp Thr 510 515 gtgaacattc tatccttgac gagcatgcca gctgagtggt agggcccacc taccagagcc 1764 aaggcctgca caaaacaaag gccacctggc tttgcagtta cttgagtttg gagccagaat 1824 gcaaggccgt gaagcaaata gcagttccgt gctgccttgc cttgccggcg agcttggcga 1884 gacccgcagc tgtagtagaa gccagttccc agcacagcta aatggcttga aaacagagga 1944 cagaaagctg agagattgct ctgcaatagg tgttgagggg ctgtcccgac aggtgactga 2004 actcactaac aacttcatct ataaatctca cccatcctgt tgtctgccaa cctgtgtgcc 2064 ttttttgtaa aatgttttcg tgtctttgaa atgcctgttg aatatctaga gtttagtacc 2124 tccttctaca aacttttttg agtctttctg gg 2156 2 517 PRT Rattus norvegicus 2 Met Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Asp Gln Leu Glu Thr 1 5 10 15 Ile Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser Asn Lys Phe Lys 20 25 30 Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 35 40 45 Gly Asn Gln Val Ser Glu Tyr Ile Ser Asn Thr Phe Leu Asp Lys Gln 50 55 60 Asn Asp Val Glu Ile Pro Ser Pro Thr Gln Lys Asp Arg Glu Lys Lys 65 70 75 80 Lys Lys Gln Gln Leu Met Thr Gln Ile Ser Gly Val Lys Lys Leu Met 85 90 95 His Ser Ser Ser Leu Asn Asn Thr Ser Ile Ser Arg Phe Gly Val Asn 100 105 110 Thr Glu Asn Glu Asp His Leu Ala Lys Glu Leu Glu Asp Leu Asn Lys 115 120 125 Trp Gly Leu Asn Ile Phe Asn Val Ala Gly Tyr Ser His Asn Arg Pro 130 135 140 Leu Thr Cys Ile Met Tyr Ala Ile Phe Gln Glu Arg Asp Leu Leu Lys 145 150 155 160 Thr Phe Lys Ile Ser Ser Asp Thr Phe Val Thr Tyr Met Met Thr Leu 165 170 175 Glu Asp His Tyr His Ser Asp Val Ala Tyr His Asn Ser Leu His Ala 180 185 190 Ala Asp Val Ala Gln Ser Thr His Val Leu Leu Ser Thr Pro Ala Leu 195 200 205 Asp Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Ile Phe Ala Ala 210 215 220 Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile 225 230 235 240 Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu 245 250 255 Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Glu His 260 265 270 Cys Asp Ile Phe Gln Asn Leu Thr Lys Lys Gln Arg Gln Thr Leu Arg 275 280 285 Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met 290 295 300 Ser Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr 305 310 315 320 Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Thr Asp Arg Ile Gln Val 325 330 335 Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Ser 340 345 350 Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Glu Glu Phe Phe 355 360 365 Gln Gln Gly Asp Lys Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met 370 375 380 Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile 385 390 395 400 Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val Gln 405 410 415 Pro Asp Ala Gln Asp Ile Leu Asp Thr Leu Glu Asp Asn Arg Asn Trp 420 425 430 Tyr Gln Ser Met Ile Pro Gln Ser Pro Ser Pro Pro Leu Asp Glu Arg 435 440 445 Ser Arg Asp Cys Gln Gly Leu Met Glu Lys Phe Gln Phe Glu Leu Thr 450 455 460 Leu Glu Glu Glu Asp Ser Glu Gly Pro Glu Lys Glu Gly Glu Gly Pro 465 470 475 480 Asn Tyr Phe Ser Ser Thr Lys Thr Leu Cys Val Ile Asp Pro Glu Asn 485 490 495 Arg Asp Ser Leu Glu Glu Thr Asp Ile Asp Ile Ala Thr Glu Asp Lys 500 505 510 Ser Leu Ile Asp Thr 515 3 22 DNA artificial sequence synthetic primer 3 ggccaaaaag gccgcggccg ca 22 4 30 DNA artificial sequence synthetic primer 4 tcgaccggtt tttccggcgc cggcgttcga 30 5 17 DNA artificial sequence synthetic primer 5 cacccgctga caaacct 17 6 18 DNA artificial sequence synthetic primer 6 atggagacgc tggaggaa 18 7 18 DNA artificial sequence synthetic primer 7 atacgccaca tcagaatg 18 8 18 DNA artificial sequence synthetic primer 8 taccagagta tgattccc 18 9 18 DNA artificial sequence synthetic primer 9 gtgtcgatca gagacttg 18 10 18 DNA artificial sequence synthetic primer 10 gcacacaggt tggcagac 18 11 2706 DNA Homo sapiens CDS (522)..(2705) 11 aagcttgcgg ccgcgcggcc taggccgcat cccggagctg caactggtgg ccttcccggt 60 ggcggtggcg gctgaggacg aggcgttcct gcccgagccc ctggccccgc gcgcgccccg 120 ccgcccgcgt tcgccgccct cctcgcccgt cttcttcgcc agcccgtccc caactttccg 180 cagacgcctt cggcttctcc gcagctgcca ggatttgggc cgccagggtt gggctggggc 240 tggcttcgag gcagagaatg ggccgacacc atctcctggc cgcagccccc tggactcgca 300 ggcgagccca ggactcgtgc tgcacgccgg ggcggccacc agccagcgcc gggagtcctt 360 cctgtaccgc tcagacagcg actatgacat gtcacccaag accatgtccc ggaactcatc 420 ggtcaccagc gaggcgcacg ctgaagacct catcgtaaca ccatttgctc aggtgctggc 480 cagcctccgg agcgtccgta gcaacttctc actcctgacc a atg tgc ccg ttc cca 536 Met Cys Pro Phe Pro 1 5 gta aca agc ggt ccc ccg ctg ggc ggc ccc acc cct gtc tgc aag gcc 584 Val Thr Ser Gly Pro Pro Leu Gly Gly Pro Thr Pro Val Cys Lys Ala 10 15 20 acg ctg tca gaa gaa acg tgt cag cag ttg gcc cgg gag act ctg gag 632 Thr Leu Ser Glu Glu Thr Cys Gln Gln Leu Ala Arg Glu Thr Leu Glu 25 30 35 gag ctg gac tgg tgt ctg gag cag ctg gag acc atg cag acc tat cgc 680 Glu Leu Asp Trp Cys Leu Glu Gln Leu Glu Thr Met Gln Thr Tyr Arg 40 45 50 tct gtc agc gag atg gcc tcg cac aag ttc aaa agg atg ttg aac cgt 728 Ser Val Ser Glu Met Ala Ser His Lys Phe Lys Arg Met Leu Asn Arg 55 60 65 gag ctc aca cac ctg tca gaa atg agc agg tcc gga aac cag gtc tca 776 Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser Gly Asn Gln Val Ser 70 75 80 85 gag tac att tcc aca aca ttc ctg gac aaa cag aat gaa gtg gag atc 824 Glu Tyr Ile Ser Thr Thr Phe Leu Asp Lys Gln Asn Glu Val Glu Ile 90 95 100 cca tca ccc acg atg aag gaa cga gaa aaa cag caa gcg ccg cga cca 872 Pro Ser Pro Thr Met Lys Glu Arg Glu Lys Gln Gln Ala Pro Arg Pro 105 110 115 aga ccc tcc cag ccg ccc ccg ccc cct gta cca cac tta cag ccc atg 920 Arg Pro Ser Gln Pro Pro Pro Pro Pro Val Pro His Leu Gln Pro Met 120 125 130 tcc caa atc aca ggg ttg aaa aag ttg atg cat agt aac agc ctg aac 968 Ser Gln Ile Thr Gly Leu Lys Lys Leu Met His Ser Asn Ser Leu Asn 135 140 145 aac tct aac att ccc cga ttt ggg gtg aag acc gat caa gaa gag ctc 1016 Asn Ser Asn Ile Pro Arg Phe Gly Val Lys Thr Asp Gln Glu Glu Leu 150 155 160 165 ctg gcc caa gaa ctg gag aac ctg aac aag tgg ggc ctg aac atc ttt 1064 Leu Ala Gln Glu Leu Glu Asn Leu Asn Lys Trp Gly Leu Asn Ile Phe 170 175 180 tgc gtg tcg gat tac gct gga ggc cgc tca ctc acc tgc atc atg tac 1112 Cys Val Ser Asp Tyr Ala Gly Gly Arg Ser Leu Thr Cys Ile Met Tyr 185 190 195 atg ata ttc cag gag cgg gac ctg ctg aag aaa ttc cgc atc cct gtg 1160 Met Ile Phe Gln Glu Arg Asp Leu Leu Lys Lys Phe Arg Ile Pro Val 200 205 210 gac acg atg gtg aca tac atg ctg acg ctg gag gat cac tac cac gct 1208 Asp Thr Met Val Thr Tyr Met Leu Thr Leu Glu Asp His Tyr His Ala 215 220 225 gac gtg gcc tac cat aac agc ctg cac gca gct gac gtg ctg cag tcc 1256 Asp Val Ala Tyr His Asn Ser Leu His Ala Ala Asp Val Leu Gln Ser 230 235 240 245 acc cac gta ctg ctg gcc acg cct tgg cca acc tta agg aat gca gtg 1304 Thr His Val Leu Leu Ala Thr Pro Trp Pro Thr Leu Arg Asn Ala Val 250 255 260 ttc acg gac ctg gag att ctc gcc gcc ctc ttc gcg gct gcc atc cac 1352 Phe Thr Asp Leu Glu Ile Leu Ala Ala Leu Phe Ala Ala Ala Ile His 265 270 275 gat gtg gat cac cct ggg gtc tcc aac cag ttc ctc atc aac acc aat 1400 Asp Val Asp His Pro Gly Val Ser Asn Gln Phe Leu Ile Asn Thr Asn 280 285 290 tcg gag ctg gcg ctc atg tac aac gat gag tcg gtg ctc gag aat cac 1448 Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser Val Leu Glu Asn His 295 300 305 cac ctg gcc gtg ggc ttc aag ctg ctg cag gag gac aac tgc gac atc 1496 His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu Asp Asn Cys Asp Ile 310 315 320 325 ttc cag aac ctc agc aag cgc cag cgg cag agc cta cgc aag atg gtc 1544 Phe Gln Asn Leu Ser Lys Arg Gln Arg Gln Ser Leu Arg Lys Met Val 330 335 340 atc gac atg gtg ctg gcc acg gac atg tcc aag cac atg acc ctc ctg 1592 Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys His Met Thr Leu Leu 345 350 355 gct gac ctg aag acc atg gtg gag acc aag aaa gtg acc agc tca ggg 1640 Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys Val Thr Ser Ser Gly 360 365 370 gtc ctc ctg cta gat aac tac tcc gac cgc atc cag gtc ctc cgg aac 1688 Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile Gln Val Leu Arg Asn 375 380 385 atg gtg cac tgt gcc gac ctc agc aac ccc acc aag ccg ctg gag ctg 1736 Met Val His Cys Ala Asp Leu Ser Asn Pro Thr Lys Pro Leu Glu Leu 390 395 400 405 tac cgc cag tgg aca gac cgc atc atg gcc gag ttc ttc cag cag ggt 1784 Tyr Arg Gln Trp Thr Asp Arg Ile Met Ala Glu Phe Phe Gln Gln Gly 410 415 420 gac cga gag cgc gag cgt ggc atg gaa atc agc ccc atg tgt gac aag 1832 Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser Pro Met Cys Asp Lys 425 430 435 cac act gcc tcc gtg gag aag tct cag gtg ggt ttt att gac tac att 1880 His Thr Ala Ser Val Glu Lys Ser Gln Val Gly Phe Ile Asp Tyr Ile 440 445 450 gtg cac cca ttg tgg gag acc tgg gcg gac ctt gtc cac cca gat gcc 1928 Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu Val His Pro Asp Ala 455 460 465 cag gag atc ttg gac act ttg gag gac aac cgg gac tgg tac tac agc 1976 Gln Glu Ile Leu Asp Thr Leu Glu Asp Asn Arg Asp Trp Tyr Tyr Ser 470 475 480 485 gcc atc cgg cag agc cca tct ccg cca ccc gag gag gag tca agg ggg 2024 Ala Ile Arg Gln Ser Pro Ser Pro Pro Pro Glu Glu Glu Ser Arg Gly 490 495 500 cca ggc cac cca ccc ctg cct gac aag ttc cag ttt gag ctg acg ctg 2072 Pro Gly His Pro Pro Leu Pro Asp Lys Phe Gln Phe Glu Leu Thr Leu 505 510 515 gag gag gaa gag gag gaa gaa ata tca atg gcc cag ata ccg tgc aca 2120 Glu Glu Glu Glu Glu Glu Glu Ile Ser Met Ala Gln Ile Pro Cys Thr 520 525 530 gcc caa gag gca ttg act gag cag gga ttg tca gga gtc gag gaa gct 2168 Ala Gln Glu Ala Leu Thr Glu Gln Gly Leu Ser Gly Val Glu Glu Ala 535 540 545 ctg gat gca acc ata gcc tgg gag gca tcc ccg gcc cag gag tcg ttg 2216 Leu Asp Ala Thr Ile Ala Trp Glu Ala Ser Pro Ala Gln Glu Ser Leu 550 555 560 565 gaa gtt atg gca cag gaa gca tcc ctg gag gcc gag ctg gag gca gtg 2264 Glu Val Met Ala Gln Glu Ala Ser Leu Glu Ala Glu Leu Glu Ala Val 570 575 580 tat ttg aca cag cag gca cag tcc aca ggc agt gca cct gtg gct ccg 2312 Tyr Leu Thr Gln Gln Ala Gln Ser Thr Gly Ser Ala Pro Val Ala Pro 585 590 595 gat gag ttc tcg tcc cgg gag gaa ttc gtg gtt gct gta agc cac agc 2360 Asp Glu Phe Ser Ser Arg Glu Glu Phe Val Val Ala Val Ser His Ser 600 605 610 agc ccc tct gcc ctg gct ctt caa agc ccc ctt ctc cct gct tgg agg 2408 Ser Pro Ser Ala Leu Ala Leu Gln Ser Pro Leu Leu Pro Ala Trp Arg 615 620 625 acc ctg tct gtt tca gag cat gcc cgg cct ccc ggg cct ccc ctc cac 2456 Thr Leu Ser Val Ser Glu His Ala Arg Pro Pro Gly Pro Pro Leu His 630 635 640 645 ggc ggc cga ggt gga ggc cca acg aga gca cca ggc tgc caa gag ggc 2504 Gly Gly Arg Gly Gly Gly Pro Thr Arg Ala Pro Gly Cys Gln Glu Gly 650 655 660 ttg cag tgc ctg cgc agg gac att tgg gga gga cac atc cgc act ccc 2552 Leu Gln Cys Leu Arg Arg Asp Ile Trp Gly Gly His Ile Arg Thr Pro 665 670 675 agc tcc tgg tgg cgg ggg gtc agg tgg aga ccc tac ctg atc ccc aga 2600 Ser Ser Trp Trp Arg Gly Val Arg Trp Arg Pro Tyr Leu Ile Pro Arg 680 685 690 cct ctg tcc ctg ttc ccc tcc act cct ccc ctc act ccc ctg ctc ccc 2648 Pro Leu Ser Leu Phe Pro Ser Thr Pro Pro Leu Thr Pro Leu Leu Pro 695 700 705 cga cca cct cct cct ctg cct caa aga ctc ttg tcc tct tgt ccg cgg 2696 Arg Pro Pro Pro Pro Leu Pro Gln Arg Leu Leu Ser Ser Cys Pro Arg 710 715 720 725 ccg caa gct t 2706 Pro Gln Ala 12 728 PRT Homo sapiens 12 Met Cys Pro Phe Pro Val Thr Ser Gly Pro Pro Leu Gly Gly Pro Thr 1 5 10 15 Pro Val Cys Lys Ala Thr Leu Ser Glu Glu Thr Cys Gln Gln Leu Ala 20 25 30 Arg Glu Thr Leu Glu Glu Leu Asp Trp Cys Leu Glu Gln Leu Glu Thr 35 40 45 Met Gln Thr Tyr Arg Ser Val Ser Glu Met Ala Ser His Lys Phe Lys 50 55 60 Arg Met Leu Asn Arg Glu Leu Thr His Leu Ser Glu Met Ser Arg Ser 65 70 75 80 Gly Asn Gln Val Ser Glu Tyr Ile Ser Thr Thr Phe Leu Asp Lys Gln 85 90 95 Asn Glu Val Glu Ile Pro Ser Pro Thr Met Lys Glu Arg Glu Lys Gln 100 105 110 Gln Ala Pro Arg Pro Arg Pro Ser Gln Pro Pro Pro Pro Pro Val Pro 115 120 125 His Leu Gln Pro Met Ser Gln Ile Thr Gly Leu Lys Lys Leu Met His 130 135 140 Ser Asn Ser Leu Asn Asn Ser Asn Ile Pro Arg Phe Gly Val Lys Thr 145 150 155 160 Asp Gln Glu Glu Leu Leu Ala Gln Glu Leu Glu Asn Leu Asn Lys Trp 165 170 175 Gly Leu Asn Ile Phe Cys Val Ser Asp Tyr Ala Gly Gly Arg Ser Leu 180 185 190 Thr Cys Ile Met Tyr Met Ile Phe Gln Glu Arg Asp Leu Leu Lys Lys 195 200 205 Phe Arg Ile Pro Val Asp Thr Met Val Thr Tyr Met Leu Thr Leu Glu 210 215 220 Asp His Tyr His Ala Asp Val Ala Tyr His Asn Ser Leu His Ala Ala 225 230 235 240 Asp Val Leu Gln Ser Thr His Val Leu Leu Ala Thr Pro Trp Pro Thr 245 250 255 Leu Arg Asn Ala Val Phe Thr Asp Leu Glu Ile Leu Ala Ala Leu Phe 260 265 270 Ala Ala Ala Ile His Asp Val Asp His Pro Gly Val Ser Asn Gln Phe 275 280 285 Leu Ile Asn Thr Asn Ser Glu Leu Ala Leu Met Tyr Asn Asp Glu Ser 290 295 300 Val Leu Glu Asn His His Leu Ala Val Gly Phe Lys Leu Leu Gln Glu 305 310 315 320 Asp Asn Cys Asp Ile Phe Gln Asn Leu Ser Lys Arg Gln Arg Gln Ser 325 330 335 Leu Arg Lys Met Val Ile Asp Met Val Leu Ala Thr Asp Met Ser Lys 340 345 350 His Met Thr Leu Leu Ala Asp Leu Lys Thr Met Val Glu Thr Lys Lys 355 360 365 Val Thr Ser Ser Gly Val Leu Leu Leu Asp Asn Tyr Ser Asp Arg Ile 370 375 380 Gln Val Leu Arg Asn Met Val His Cys Ala Asp Leu Ser Asn Pro Thr 385 390 395 400 Lys Pro Leu Glu Leu Tyr Arg Gln Trp Thr Asp Arg Ile Met Ala Glu 405 410 415 Phe Phe Gln Gln Gly Asp Arg Glu Arg Glu Arg Gly Met Glu Ile Ser 420 425 430 Pro Met Cys Asp Lys His Thr Ala Ser Val Glu Lys Ser Gln Val Gly 435 440 445 Phe Ile Asp Tyr Ile Val His Pro Leu Trp Glu Thr Trp Ala Asp Leu 450 455 460 Val His Pro Asp Ala Gln Glu Ile Leu Asp Thr Leu Glu Asp Asn Arg 465 470 475 480 Asp Trp Tyr Tyr Ser Ala Ile Arg Gln Ser Pro Ser Pro Pro Pro Glu 485 490 495 Glu Glu Ser Arg Gly Pro Gly His Pro Pro Leu Pro Asp Lys Phe Gln 500 505 510 Phe Glu Leu Thr Leu Glu Glu Glu Glu Glu Glu Glu Ile Ser Met Ala 515 520 525 Gln Ile Pro Cys Thr Ala Gln Glu Ala Leu Thr Glu Gln Gly Leu Ser 530 535 540 Gly Val Glu Glu Ala Leu Asp Ala Thr Ile Ala Trp Glu Ala Ser Pro 545 550 555 560 Ala Gln Glu Ser Leu Glu Val Met Ala Gln Glu Ala Ser Leu Glu Ala 565 570 575 Glu Leu Glu Ala Val Tyr Leu Thr Gln Gln Ala Gln Ser Thr Gly Ser 580 585 590 Ala Pro Val Ala Pro Asp Glu Phe Ser Ser Arg Glu Glu Phe Val Val 595 600 605 Ala Val Ser His Ser Ser Pro Ser Ala Leu Ala Leu Gln Ser Pro Leu 610 615 620 Leu Pro Ala Trp Arg Thr Leu Ser Val Ser Glu His Ala Arg Pro Pro 625 630 635 640 Gly Pro Pro Leu His Gly Gly Arg Gly Gly Gly Pro Thr Arg Ala Pro 645 650 655 Gly Cys Gln Glu Gly Leu Gln Cys Leu Arg Arg Asp Ile Trp Gly Gly 660 665 670 His Ile Arg Thr Pro Ser Ser Trp Trp Arg Gly Val Arg Trp Arg Pro 675 680 685 Tyr Leu Ile Pro Arg Pro Leu Ser Leu Phe Pro Ser Thr Pro Pro Leu 690 695 700 Thr Pro Leu Leu Pro Arg Pro Pro Pro Pro Leu Pro Gln Arg Leu Leu 705 710 715 720 Ser Ser Cys Pro Arg Pro Gln Ala 725 13 1721 DNA Homo sapiens CDS (60)..(1274) 13 aagcttgcgg ccgcattggg taccgcgtgc cagcaggcag tggccctagc cttccgcct 59 atg ccc tcc ctc caa gag gtg gac tgc ggc tcc ccc agc agc tcc gag 107 Met Pro Ser Leu Gln Glu Val Asp Cys Gly Ser Pro Ser Ser Ser Glu 1 5 10 15 gag gag ggg gtg cca ggg tcc cgg ggg agc cca gcg acc tca ccc cac 155 Glu Glu Gly Val Pro Gly Ser Arg Gly Ser Pro Ala Thr Ser Pro His 20 25 30 ctg ggc cgc cga cga cct ctg ctt cgg tcc atg agc gcc gcc ttc tgc 203 Leu Gly Arg Arg Arg Pro Leu Leu Arg Ser Met Ser Ala Ala Phe Cys 35 40 45 tcc cta ctg gca ccg gag cgg cag gtg ggc cgg gct gcg gca gca ctg 251 Ser Leu Leu Ala Pro Glu Arg Gln Val Gly Arg Ala Ala Ala Ala Leu 50 55 60 atg cag gac cga cac aca gcc gcg ggc cag ctg gtg cag gac cta ctg 299 Met Gln Asp Arg His Thr Ala Ala Gly Gln Leu Val Gln Asp Leu Leu 65 70 75 80 acc cag gtg cgg gat ggg cag agg ccc cag gag ctc gag ggc atc cgt 347 Thr Gln Val Arg Asp Gly Gln Arg Pro Gln Glu Leu Glu Gly Ile Arg 85 90 95 cag gcg ctg agc cgg gcc cgg gcc atg ctg agt gcg gag ctg ggc cct 395 Gln Ala Leu Ser Arg Ala Arg Ala Met Leu Ser Ala Glu Leu Gly Pro 100 105 110 gag aag ctc gtg tcg cct aag agg ctg gaa cat gtc ctg gag aag tca 443 Glu Lys Leu Val Ser Pro Lys Arg Leu Glu His Val Leu Glu Lys Ser 115 120 125 ttg cat tgc tct gtg ctc aag cct ctc cgg ccc atc ctg gca gcc cgc 491 Leu His Cys Ser Val Leu Lys Pro Leu Arg Pro Ile Leu Ala Ala Arg 130 135 140 ctg cgg cgc cgg ctt gcc gca gac ggc tcc ctg ggc cgc cta gct gag 539 Leu Arg Arg Arg Leu Ala Ala Asp Gly Ser Leu Gly Arg Leu Ala Glu 145 150 155 160 ggc ctc cgc ctg gcc cgg gcc cag ggc ccc gga gcc ttc ggg tcc cac 587 Gly Leu Arg Leu Ala Arg Ala Gln Gly Pro Gly Ala Phe Gly Ser His 165 170 175 ctg agc ctg ccc tcc cca gta gag ttg gag caa gtg cgc cag aag ctg 635 Leu Ser Leu Pro Ser Pro Val Glu Leu Glu Gln Val Arg Gln Lys Leu 180 185 190 ctg cag ctc gtc cgc acc tac tca ccc agc gcc cag gtc aag cgg ctc 683 Leu Gln Leu Val Arg Thr Tyr Ser Pro Ser Ala Gln Val Lys Arg Leu 195 200 205 ctg cag gcc tgc aag ctg ctc tac atg gcc ctg agg acc cag gaa ggg 731 Leu Gln Ala Cys Lys Leu Leu Tyr Met Ala Leu Arg Thr Gln Glu Gly 210 215 220 gag ggc tcg ggt gcc gac ggg ttc ctg cct ctg ctg agc ctc gtc ttg 779 Glu Gly Ser Gly Ala Asp Gly Phe Leu Pro Leu Leu Ser Leu Val Leu 225 230 235 240 gcc cac tgt gac ctt cct gag ctg ctg ctg gag gcc gag tac atg tcg 827 Ala His Cys Asp Leu Pro Glu Leu Leu Leu Glu Ala Glu Tyr Met Ser 245 250 255 gag ctg ctg gag ccc agc ctg ctt act gga gag ggt ggc tac tac ctg 875 Glu Leu Leu Glu Pro Ser Leu Leu Thr Gly Glu Gly Gly Tyr Tyr Leu 260 265 270 acc agc ctc tct gcc agc ctg gcc ctg ctg agt ggc ctg ggt cag gcc 923 Thr Ser Leu Ser Ala Ser Leu Ala Leu Leu Ser Gly Leu Gly Gln Ala 275 280 285 cac acc ctc cca ctg agc ccc gtg cag gag cta cgg cgc tcc ctc agc 971 His Thr Leu Pro Leu Ser Pro Val Gln Glu Leu Arg Arg Ser Leu Ser 290 295 300 ctc tgg gag cag cgc cgc ctg cct gcc acc cac tgc ttc cag cac ctc 1019 Leu Trp Glu Gln Arg Arg Leu Pro Ala Thr His Cys Phe Gln His Leu 305 310 315 320 ctc cga gta gcc tat cag gat ccc agc agt ggc tgc acc tcc aag acc 1067 Leu Arg Val Ala Tyr Gln Asp Pro Ser Ser Gly Cys Thr Ser Lys Thr 325 330 335 ctg gcc gtg ccc cca gag gcc tcg att gcc acc ctg aac cag ctc tgt 1115 Leu Ala Val Pro Pro Glu Ala Ser Ile Ala Thr Leu Asn Gln Leu Cys 340 345 350 gcc acc aag ttc cga gtg acc cag ccc aac act ttt ggc ctc ttc ctg 1163 Ala Thr Lys Phe Arg Val Thr Gln Pro Asn Thr Phe Gly Leu Phe Leu 355 360 365 tac aag gag cag ggc tac cac cgc ctg ccc cct ggg ccc tgg ccc aca 1211 Tyr Lys Glu Gln Gly Tyr His Arg Leu Pro Pro Gly Pro Trp Pro Thr 370 375 380 ggc tgc cca cca ctg gct acc tcg tct acc gcc ggg cag agt ggc ctg 1259 Gly Cys Pro Pro Leu Ala Thr Ser Ser Thr Ala Gly Gln Ser Gly Leu 385 390 395 400 aga ccc agg ggg ctg tgacagagga ggagggcagt gggcagtcag aggcaagaag 1314 Arg Pro Arg Gly Leu 405 cagaggggag gagcaagggt gccagggaga tggggatgct ggggtcaaag ccagccccag 1374 ggacattcgg gaacagtctg agacaactgc tgaagggggc cagggtcaag cccaggaagg 1434 ccctgctcag ccaggggaac cagaggcaga gggaagccgg gcagcagagg agtagcttga 1494 agtggccaga agggtcattc ggggcgggag accctgagcc tgctgagaaa tccttttagc 1554 gccagcaagc cccacccagg gccctgtcct gtgtctgcca ccacctttgt ctgatacttg 1614 tttccaggga agctggggga actgccacat ctgaggaact ggaataaaga tgaggggcct 1674 tcgggggcca atgcggccgc cgcggccttt ttggccagct cgaattc 1721 14 405 PRT Homo sapiens 14 Met Pro Ser Leu Gln Glu Val Asp Cys Gly Ser Pro Ser Ser Ser Glu 1 5 10 15 Glu Glu Gly Val Pro Gly Ser Arg Gly Ser Pro Ala Thr Ser Pro His 20 25 30 Leu Gly Arg Arg Arg Pro Leu Leu Arg Ser Met Ser Ala Ala Phe Cys 35 40 45 Ser Leu Leu Ala Pro Glu Arg Gln Val Gly Arg Ala Ala Ala Ala Leu 50 55 60 Met Gln Asp Arg His Thr Ala Ala Gly Gln Leu Val Gln Asp Leu Leu 65 70 75 80 Thr Gln Val Arg Asp Gly Gln Arg Pro Gln Glu Leu Glu Gly Ile Arg 85 90 95 Gln Ala Leu Ser Arg Ala Arg Ala Met Leu Ser Ala Glu Leu Gly Pro 100 105 110 Glu Lys Leu Val Ser Pro Lys Arg Leu Glu His Val Leu Glu Lys Ser 115 120 125 Leu His Cys Ser Val Leu Lys Pro Leu Arg Pro Ile Leu Ala Ala Arg 130 135 140 Leu Arg Arg Arg Leu Ala Ala Asp Gly Ser Leu Gly Arg Leu Ala Glu 145 150 155 160 Gly Leu Arg Leu Ala Arg Ala Gln Gly Pro Gly Ala Phe Gly Ser His 165 170 175 Leu Ser Leu Pro Ser Pro Val Glu Leu Glu Gln Val Arg Gln Lys Leu 180 185 190 Leu Gln Leu Val Arg Thr Tyr Ser Pro Ser Ala Gln Val Lys Arg Leu 195 200 205 Leu Gln Ala Cys Lys Leu Leu Tyr Met Ala Leu Arg Thr Gln Glu Gly 210 215 220 Glu Gly Ser Gly Ala Asp Gly Phe Leu Pro Leu Leu Ser Leu Val Leu 225 230 235 240 Ala His Cys Asp Leu Pro Glu Leu Leu Leu Glu Ala Glu Tyr Met Ser 245 250 255 Glu Leu Leu Glu Pro Ser Leu Leu Thr Gly Glu Gly Gly Tyr Tyr Leu 260 265 270 Thr Ser Leu Ser Ala Ser Leu Ala Leu Leu Ser Gly Leu Gly Gln Ala 275 280 285 His Thr Leu Pro Leu Ser Pro Val Gln Glu Leu Arg Arg Ser Leu Ser 290 295 300 Leu Trp Glu Gln Arg Arg Leu Pro Ala Thr His Cys Phe Gln His Leu 305 310 315 320 Leu Arg Val Ala Tyr Gln Asp Pro Ser Ser Gly Cys Thr Ser Lys Thr 325 330 335 Leu Ala Val Pro Pro Glu Ala Ser Ile Ala Thr Leu Asn Gln Leu Cys 340 345 350 Ala Thr Lys Phe Arg Val Thr Gln Pro Asn Thr Phe Gly Leu Phe Leu 355 360 365 Tyr Lys Glu Gln Gly Tyr His Arg Leu Pro Pro Gly Pro Trp Pro Thr 370 375 380 Gly Cys Pro Pro Leu Ala Thr Ser Ser Thr Ala Gly Gln Ser Gly Leu 385 390 395 400 Arg Pro Arg Gly Leu 405 15 1829 DNA Homo sapiens CDS (30)..(1421) 15 gcggccgcgg ccggcagcgg ctgagcgac atg agc att tct act tcc tcc tcc 53 Met Ser Ile Ser Thr Ser Ser Ser 1 5 gac tcg ctg gag ttc gac cgg agc atg cct ctg ttt ggc tac gag gcg 101 Asp Ser Leu Glu Phe Asp Arg Ser Met Pro Leu Phe Gly Tyr Glu Ala 10 15 20 gac acc aac agc agc ctg gag gac tac gag ggg gaa agt gac caa gag 149 Asp Thr Asn Ser Ser Leu Glu Asp Tyr Glu Gly Glu Ser Asp Gln Glu 25 30 35 40 acc atg gcg ccc ccc atc aag tcc aaa aag aaa agg agc agc tcc ttc 197 Thr Met Ala Pro Pro Ile Lys Ser Lys Lys Lys Arg Ser Ser Ser Phe 45 50 55 gtg ctg ccc aag ctc gtc aag tcc cag ctg cag aag gtg agc ggg gtg 245 Val Leu Pro Lys Leu Val Lys Ser Gln Leu Gln Lys Val Ser Gly Val 60 65 70 ttc agc tcc ttc atg acc ccg gag aag cgg atg gtc cgc agg atc gcc 293 Phe Ser Ser Phe Met Thr Pro Glu Lys Arg Met Val Arg Arg Ile Ala 75 80 85 gag ctt tcc cgg gac aaa tgc acc tac ttc ggg tgc tta gtg cag gac 341 Glu Leu Ser Arg Asp Lys Cys Thr Tyr Phe Gly Cys Leu Val Gln Asp 90 95 100 tac gtg agc ttc ctg cag gag aac aag gag tgc cac gtg tcc agc acc 389 Tyr Val Ser Phe Leu Gln Glu Asn Lys Glu Cys His Val Ser Ser Thr 105 110 115 120 gac atg ctg cag acc atc cgg cag ttc atg acc cag gtc aag aac tat 437 Asp Met Leu Gln Thr Ile Arg Gln Phe Met Thr Gln Val Lys Asn Tyr 125 130 135 ttg tct cag agc tcg gag ctg gac ccc ccc atc gag tcg ctg atc cct 485 Leu Ser Gln Ser Ser Glu Leu Asp Pro Pro Ile Glu Ser Leu Ile Pro 140 145 150 gaa gac caa ata gat gtg gtg ctg gaa aaa gcc atg cac aag tgc atc 533 Glu Asp Gln Ile Asp Val Val Leu Glu Lys Ala Met His Lys Cys Ile 155 160 165 ttg aag ccc ctc aag ggg cac gtg gag gcc atg ctg aag gac ttt cac 581 Leu Lys Pro Leu Lys Gly His Val Glu Ala Met Leu Lys Asp Phe His 170 175 180 atg gcc gat ggc tca tgg aag caa ctc aag gag aac ctg cag ctt gtg 629 Met Ala Asp Gly Ser Trp Lys Gln Leu Lys Glu Asn Leu Gln Leu Val 185 190 195 200 cgg cag agg aat ccg cag gag ctg ggg gtc ttc gcc ccg acc cct gat 677 Arg Gln Arg Asn Pro Gln Glu Leu Gly Val Phe Ala Pro Thr Pro Asp 205 210 215 ttt gtg gat gtg gag aaa atc aaa gtc aag ttc atg acc atg cag aag 725 Phe Val Asp Val Glu Lys Ile Lys Val Lys Phe Met Thr Met Gln Lys 220 225 230 atg tat tcg ccg gaa aag aag gtc atg ctg ctg ctg cgg gtc tgc aag 773 Met Tyr Ser Pro Glu Lys Lys Val Met Leu Leu Leu Arg Val Cys Lys 235 240 245 ctc att tac acg gtc atg gag aac aac tca ggg agg atg tat ggc gct 821 Leu Ile Tyr Thr Val Met Glu Asn Asn Ser Gly Arg Met Tyr Gly Ala 250 255 260 gat gac ttc ttg cca gtc ctg acc tat gtc ata gcc cag tgt gac atg 869 Asp Asp Phe Leu Pro Val Leu Thr Tyr Val Ile Ala Gln Cys Asp Met 265 270 275 280 ctt gaa ttg gac act gaa atc gag tac atg atg gag ctc cta gac cca 917 Leu Glu Leu Asp Thr Glu Ile Glu Tyr Met Met Glu Leu Leu Asp Pro 285 290 295 tcg ctg tta cat gga gaa gga ggc tat tac ttg aca agc gca tat gga 965 Ser Leu Leu His Gly Glu Gly Gly Tyr Tyr Leu Thr Ser Ala Tyr Gly 300 305 310 gca ctt tct ctg ata aag aat ttc caa gaa gaa caa gca gcg cga ctg 1013 Ala Leu Ser Leu Ile Lys Asn Phe Gln Glu Glu Gln Ala Ala Arg Leu 315 320 325 ctc agc tca gaa acc aga gac acc ctg agg cag tgg cac aaa cgg aga 1061 Leu Ser Ser Glu Thr Arg Asp Thr Leu Arg Gln Trp His Lys Arg Arg 330 335 340 acc acc aac cgg acc atc ccc tct gtg gac gac ttc cag aat tac ctc 1109 Thr Thr Asn Arg Thr Ile Pro Ser Val Asp Asp Phe Gln Asn Tyr Leu 345 350 355 360 cga gtt gca ttt cag gag gtc aac agt ggt tgc aca gga aag acc ctc 1157 Arg Val Ala Phe Gln Glu Val Asn Ser Gly Cys Thr Gly Lys Thr Leu 365 370 375 ctt gtg aga cct tac atc acc act gag gat gtg tgt cag atc tgc gct 1205 Leu Val Arg Pro Tyr Ile Thr Thr Glu Asp Val Cys Gln Ile Cys Ala 380 385 390 gag aag ttc aag gtg ggg gac cct gag gag tac agc ctc ttt ctc ttc 1253 Glu Lys Phe Lys Val Gly Asp Pro Glu Glu Tyr Ser Leu Phe Leu Phe 395 400 405 gtt gac gag aca tgg cag cag ctg gca gag gac act tac cct caa aaa 1301 Val Asp Glu Thr Trp Gln Gln Leu Ala Glu Asp Thr Tyr Pro Gln Lys 410 415 420 atc aag gcg gag ctg cac agc cga cca cag ccc cac atc ttc cac ttt 1349 Ile Lys Ala Glu Leu His Ser Arg Pro Gln Pro His Ile Phe His Phe 425 430 435 440 gtc tac aaa cgc atc aag aac gat cct tat ggc atc att ttc cag aac 1397 Val Tyr Lys Arg Ile Lys Asn Asp Pro Tyr Gly Ile Ile Phe Gln Asn 445 450 455 ggg gaa gaa gac ctc acc acc tcc tagaagacag gcgggacttc ccagtggtgc 1451 Gly Glu Glu Asp Leu Thr Thr Ser 460 atccaaaggg gagctggaag ccttgccttc ccgcttctac atgcttgagc ttgaaaagca 1511 gtcacctcct cggggacccc tcagtgtagt gactaagcca tccacaggcc aactcggcca 1571 agggcaactt tagccacgca aggtagctga ggtttgtgaa acagtaggat tctcttttgg 1631 caatggagaa ttgcatctga tggttcaagt gtcctgagat tgtttgctac ctacccccag 1691 tcaggttcta ggttggctta caggtatgta tatgtgcaga agaaacactt aagatacaag 1751 ttcttttgaa ttcaacagca gatgcttgcg atgcagtgcg tcaggtgatt ctcactcctg 1811 tggatggctt catccctg 1829 16 464 PRT Homo sapiens 16 Met Ser Ile Ser Thr Ser Ser Ser Asp Ser Leu Glu Phe Asp Arg Ser 1 5 10 15 Met Pro Leu Phe Gly Tyr Glu Ala Asp Thr Asn Ser Ser Leu Glu Asp 20 25 30 Tyr Glu Gly Glu Ser Asp Gln Glu Thr Met Ala Pro Pro Ile Lys Ser 35 40 45 Lys Lys Lys Arg Ser Ser Ser Phe Val Leu Pro Lys Leu Val Lys Ser 50 55 60 Gln Leu Gln Lys Val Ser Gly Val Phe Ser Ser Phe Met Thr Pro Glu 65 70 75 80 Lys Arg Met Val Arg Arg Ile Ala Glu Leu Ser Arg Asp Lys Cys Thr 85 90 95 Tyr Phe Gly Cys Leu Val Gln Asp Tyr Val Ser Phe Leu Gln Glu Asn 100 105 110 Lys Glu Cys His Val Ser Ser Thr Asp Met Leu Gln Thr Ile Arg Gln 115 120 125 Phe Met Thr Gln Val Lys Asn Tyr Leu Ser Gln Ser Ser Glu Leu Asp 130 135 140 Pro Pro Ile Glu Ser Leu Ile Pro Glu Asp Gln Ile Asp Val Val Leu 145 150 155 160 Glu Lys Ala Met His Lys Cys Ile Leu Lys Pro Leu Lys Gly His Val 165 170 175 Glu Ala Met Leu Lys Asp Phe His Met Ala Asp Gly Ser Trp Lys Gln 180 185 190 Leu Lys Glu Asn Leu Gln Leu Val Arg Gln Arg Asn Pro Gln Glu Leu 195 200 205 Gly Val Phe Ala Pro Thr Pro Asp Phe Val Asp Val Glu Lys Ile Lys 210 215 220 Val Lys Phe Met Thr Met Gln Lys Met Tyr Ser Pro Glu Lys Lys Val 225 230 235 240 Met Leu Leu Leu Arg Val Cys Lys Leu Ile Tyr Thr Val Met Glu Asn 245 250 255 Asn Ser Gly Arg Met Tyr Gly Ala Asp Asp Phe Leu Pro Val Leu Thr 260 265 270 Tyr Val Ile Ala Gln Cys Asp Met Leu Glu Leu Asp Thr Glu Ile Glu 275 280 285 Tyr Met Met Glu Leu Leu Asp Pro Ser Leu Leu His Gly Glu Gly Gly 290 295 300 Tyr Tyr Leu Thr Ser Ala Tyr Gly Ala Leu Ser Leu Ile Lys Asn Phe 305 310 315 320 Gln Glu Glu Gln Ala Ala Arg Leu Leu Ser Ser Glu Thr Arg Asp Thr 325 330 335 Leu Arg Gln Trp His Lys Arg Arg Thr Thr Asn Arg Thr Ile Pro Ser 340 345 350 Val Asp Asp Phe Gln Asn Tyr Leu Arg Val Ala Phe Gln Glu Val Asn 355 360 365 Ser Gly Cys Thr Gly Lys Thr Leu Leu Val Arg Pro Tyr Ile Thr Thr 370 375 380 Glu Asp Val Cys Gln Ile Cys Ala Glu Lys Phe Lys Val Gly Asp Pro 385 390 395 400 Glu Glu Tyr Ser Leu Phe Leu Phe Val Asp Glu Thr Trp Gln Gln Leu 405 410 415 Ala Glu Asp Thr Tyr Pro Gln Lys Ile Lys Ala Glu Leu His Ser Arg 420 425 430 Pro Gln Pro His Ile Phe His Phe Val Tyr Lys Arg Ile Lys Asn Asp 435 440 445 Pro Tyr Gly Ile Ile Phe Gln Asn Gly Glu Glu Asp Leu Thr Thr Ser 450 455 460 17 1299 DNA Homo sapiens CDS (1)..(1299) 17 ggc cgc att gcc gac ccg gcc cgt agt gtg gaa gca gct tca gct caa 48 Gly Arg Ile Ala Asp Pro Ala Arg Ser Val Glu Ala Ala Ser Ala Gln 1 5 10 15 aga tta gaa cga ctc cga aaa gag aga caa aac cag atc aaa tgc aaa 96 Arg Leu Glu Arg Leu Arg Lys Glu Arg Gln Asn Gln Ile Lys Cys Lys 20 25 30 aat att cag tgg aaa gaa aga aat tct aag caa tca gcc cag gag tta 144 Asn Ile Gln Trp Lys Glu Arg Asn Ser Lys Gln Ser Ala Gln Glu Leu 35 40 45 aag tca ctg ttt gaa aaa aaa tct ctc aaa gag aag cct cca att tct 192 Lys Ser Leu Phe Glu Lys Lys Ser Leu Lys Glu Lys Pro Pro Ile Ser 50 55 60 ggg aag cag tcg ata tta tct gta cgc cta gaa cag tgc cct ctg cag 240 Gly Lys Gln Ser Ile Leu Ser Val Arg Leu Glu Gln Cys Pro Leu Gln 65 70 75 80 ctg aat aac cct ttt aac gag tat tcc aaa ttt gat ggc aag ggt cat 288 Leu Asn Asn Pro Phe Asn Glu Tyr Ser Lys Phe Asp Gly Lys Gly His 85 90 95 gta ggt aca aca gca acc aag aag atc gat gtc tac ctc cct ctg cac 336 Val Gly Thr Thr Ala Thr Lys Lys Ile Asp Val Tyr Leu Pro Leu His 100 105 110 tcg agc cag gac aga ctg ctg cca atg acc gtg gtg aca atg gcc agc 384 Ser Ser Gln Asp Arg Leu Leu Pro Met Thr Val Val Thr Met Ala Ser 115 120 125 gcc agg gtg cag gac ctg atc ggg ctc atc tgc tgg cag tat aca agc 432 Ala Arg Val Gln Asp Leu Ile Gly Leu Ile Cys Trp Gln Tyr Thr Ser 130 135 140 gaa gga cgg gag ccg aag ctc aat gac aat gtc agt gcc tac tgc ctg 480 Glu Gly Arg Glu Pro Lys Leu Asn Asp Asn Val Ser Ala Tyr Cys Leu 145 150 155 160 cat att gct gag gat gat ggg gag gtg gac acc gat ttc ccc ccg ctg 528 His Ile Ala Glu Asp Asp Gly Glu Val Asp Thr Asp Phe Pro Pro Leu 165 170 175 gat tcc aat gag ccc att cat aag ttt ggc ttc agt act ttg gcc ctg 576 Asp Ser Asn Glu Pro Ile His Lys Phe Gly Phe Ser Thr Leu Ala Leu 180 185 190 gtt gaa aag tac tca tct cct ggt ctg aca tcc aaa gag tca ctc ttt 624 Val Glu Lys Tyr Ser Ser Pro Gly Leu Thr Ser Lys Glu Ser Leu Phe 195 200 205 gtt cga ata aat gct gct cat gga ttc tcc ctt att cag gtg gac aac 672 Val Arg Ile Asn Ala Ala His Gly Phe Ser Leu Ile Gln Val Asp Asn 210 215 220 aca aag gtt acc atg aag gaa atc tta ctg aag gca gtg aag cga aga 720 Thr Lys Val Thr Met Lys Glu Ile Leu Leu Lys Ala Val Lys Arg Arg 225 230 235 240 aaa gga tcc cag aaa gtt tca ggc cct cag tac cgc ctg gag aag cag 768 Lys Gly Ser Gln Lys Val Ser Gly Pro Gln Tyr Arg Leu Glu Lys Gln 245 250 255 agc gag ccc aat gtc gcc gtt gac ctg gac agc act ttg gag agc cag 816 Ser Glu Pro Asn Val Ala Val Asp Leu Asp Ser Thr Leu Glu Ser Gln 260 265 270 agc gca tgg gag ttc tgc ctg gtc cgc gag aac agt tca agg gca gac 864 Ser Ala Trp Glu Phe Cys Leu Val Arg Glu Asn Ser Ser Arg Ala Asp 275 280 285 ggg gtt ttt gag gag gat tcg caa att gac ata gcc aca gta cag gat 912 Gly Val Phe Glu Glu Asp Ser Gln Ile Asp Ile Ala Thr Val Gln Asp 290 295 300 atg ctt agc agc cac cat tac aag tca ttc aaa gtc agc atg atc cac 960 Met Leu Ser Ser His His Tyr Lys Ser Phe Lys Val Ser Met Ile His 305 310 315 320 aga ctg cga ttc aca acc gac gta cag cta ggt atc tct gga gac aaa 1008 Arg Leu Arg Phe Thr Thr Asp Val Gln Leu Gly Ile Ser Gly Asp Lys 325 330 335 gta gag ata gac cct gtt acg aat cag aaa gcc agc act aag ttt tgg 1056 Val Glu Ile Asp Pro Val Thr Asn Gln Lys Ala Ser Thr Lys Phe Trp 340 345 350 att aag cag aaa ccc atc tca atc gat tcc gac ctg ctc tgt gcc tgt 1104 Ile Lys Gln Lys Pro Ile Ser Ile Asp Ser Asp Leu Leu Cys Ala Cys 355 360 365 gac ctt gct gaa gag aaa agc ccc agt cac gca ata ttt aaa ctc acg 1152 Asp Leu Ala Glu Glu Lys Ser Pro Ser His Ala Ile Phe Lys Leu Thr 370 375 380 tat cta agc aat cac gac tat aaa cac ctc tac ttt gaa tcg gac gct 1200 Tyr Leu Ser Asn His Asp Tyr Lys His Leu Tyr Phe Glu Ser Asp Ala 385 390 395 400 gct acc gtc aat gaa att gtg ctc aag gtt aac tac atc ctg gaa tcg 1248 Ala Thr Val Asn Glu Ile Val Leu Lys Val Asn Tyr Ile Leu Glu Ser 405 410 415 cga gct agc act gcc cgg gct gac tac ttt gct caa aaa aaa agc ggc 1296 Arg Ala Ser Thr Ala Arg Ala Asp Tyr Phe Ala Gln Lys Lys Ser Gly 420 425 430 cgc 1299 Arg 18 433 PRT Homo sapiens 18 Gly Arg Ile Ala Asp Pro Ala Arg Ser Val Glu Ala Ala Ser Ala Gln 1 5 10 15 Arg Leu Glu Arg Leu Arg Lys Glu Arg Gln Asn Gln Ile Lys Cys Lys 20 25 30 Asn Ile Gln Trp Lys Glu Arg Asn Ser Lys Gln Ser Ala Gln Glu Leu 35 40 45 Lys Ser Leu Phe Glu Lys Lys Ser Leu Lys Glu Lys Pro Pro Ile Ser 50 55 60 Gly Lys Gln Ser Ile Leu Ser Val Arg Leu Glu Gln Cys Pro Leu Gln 65 70 75 80 Leu Asn Asn Pro Phe Asn Glu Tyr Ser Lys Phe Asp Gly Lys Gly His 85 90 95 Val Gly Thr Thr Ala Thr Lys Lys Ile Asp Val Tyr Leu Pro Leu His 100 105 110 Ser Ser Gln Asp Arg Leu Leu Pro Met Thr Val Val Thr Met Ala Ser 115 120 125 Ala Arg Val Gln Asp Leu Ile Gly Leu Ile Cys Trp Gln Tyr Thr Ser 130 135 140 Glu Gly Arg Glu Pro Lys Leu Asn Asp Asn Val Ser Ala Tyr Cys Leu 145 150 155 160 His Ile Ala Glu Asp Asp Gly Glu Val Asp Thr Asp Phe Pro Pro Leu 165 170 175 Asp Ser Asn Glu Pro Ile His Lys Phe Gly Phe Ser Thr Leu Ala Leu 180 185 190 Val Glu Lys Tyr Ser Ser Pro Gly Leu Thr Ser Lys Glu Ser Leu Phe 195 200 205 Val Arg Ile Asn Ala Ala His Gly Phe Ser Leu Ile Gln Val Asp Asn 210 215 220 Thr Lys Val Thr Met Lys Glu Ile Leu Leu Lys Ala Val Lys Arg Arg 225 230 235 240 Lys Gly Ser Gln Lys Val Ser Gly Pro Gln Tyr Arg Leu Glu Lys Gln 245 250 255 Ser Glu Pro Asn Val Ala Val Asp Leu Asp Ser Thr Leu Glu Ser Gln 260 265 270 Ser Ala Trp Glu Phe Cys Leu Val Arg Glu Asn Ser Ser Arg Ala Asp 275 280 285 Gly Val Phe Glu Glu Asp Ser Gln Ile Asp Ile Ala Thr Val Gln Asp 290 295 300 Met Leu Ser Ser His His Tyr Lys Ser Phe Lys Val Ser Met Ile His 305 310 315 320 Arg Leu Arg Phe Thr Thr Asp Val Gln Leu Gly Ile Ser Gly Asp Lys 325 330 335 Val Glu Ile Asp Pro Val Thr Asn Gln Lys Ala Ser Thr Lys Phe Trp 340 345 350 Ile Lys Gln Lys Pro Ile Ser Ile Asp Ser Asp Leu Leu Cys Ala Cys 355 360 365 Asp Leu Ala Glu Glu Lys Ser Pro Ser His Ala Ile Phe Lys Leu Thr 370 375 380 Tyr Leu Ser Asn His Asp Tyr Lys His Leu Tyr Phe Glu Ser Asp Ala 385 390 395 400 Ala Thr Val Asn Glu Ile Val Leu Lys Val Asn Tyr Ile Leu Glu Ser 405 410 415 Arg Ala Ser Thr Ala Arg Ala Asp Tyr Phe Ala Gln Lys Lys Ser Gly 420 425 430 Arg 19 18 DNA artificial sequence synthetic primer 19 tctaaaccgt ggaataat 18 20 26 DNA artificial sequence synthetic primer 20 gtcaaagctt cgtagaagat aacacc 26 

1. A method of cloning, in a genetically altered microorganism, a mammalian gene which is capable of modifying a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: a) providing mammalian cDNA in an expression vector capable of expressing the mammalian cDNA in the genetically altered microorganism; b) introducing the expression vector into the genetically altered microorganism, thereby producing genetically altered microorganisms containing the expression vector; c) maintaining genetically altered microorganisms containing the expression vector under conditions appropriate for growth of genetically altered microorganisms; and d) identifying genetically altered microorganisms in which the phenotypic alteration associated with the genetic alteration in the microorganism is modified.
 2. The method of claim 1 further comprising transforming E. coli with cDNA present in genetically altered microorganisms of step (d).
 3. A method of cloning a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) providing mammalian cDNA in an expression vector capable of expressing the mammalian gene in yeast; b) introducing the expression vector of step (a) into genetically altered yeast cells, thereby producing genetically altered yeast cells containing the expression vector; c) maintaining genetically altered yeast cells containing the expression vector under conditions appropriate for growth of genetically altered yeast cells; and d) identifying genetically altered yeast cells in which the phenotypic alteration associated with the genetic alteration in the yeast cell is modified.
 4. The method of claim 3 further comprising transforming E. coli with cDNA present in genetically altered yeast cells of step (d).
 5. The method of claim 3 wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.
 6. The method of claim 3 wherein the genetic alteration is a mutation in a gene encoding a RAS protein.
 7. The method of claim 6 wherein the genetic alteration is an activated RAS2^(va119) gene of S. cerevisiae.
 8. The method of claim 7 wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate and failure to sporulate.
 9. The method of claim 3 wherein the genetically altered yeast cells are genetically altered S. cerevisiae or genetically altered S. pombe.
 10. The method of claim 5 wherein the genetic alteration is a disruption of the PDE1 and of the PDE2 genes of S. cerevisiae.
 11. The method of claim 6 wherein the genetic alteration is a disruption of the ras1 gene of S. pombe or an activated allele of the ras1 gene of S. pombe.
 12. Isolated DNA encoding a cAMP phosphodiesterase obtained by the method of claim
 1. 13. Isolated DNA having the nucleotide sequence of FIG.
 4. 14. Isolated protein encoded by the DNA of claim
 13. 15. Isolated DNA having the nucleotide sequence of FIG.
 5. 16. Isolated protein encoded by the DNA of claim
 15. 17. Isolated DNA having the nucleotide sequence of FIG.
 6. 18. Isolated protein encoded by the DNA of claim
 17. 19. Isolated DNA having the nucleotide sequence of FIG.
 7. 20. Isolated protein encoded by the DNA of claim
 19. 21. Isolated protein encoded by a nucleotide sequence which hybridizes to DNA having a nucleotide sequence selected from the group consisting of: a) the nucleotide sequence of FIG. 4; b) the nucleotide sequence of FIG. 5; c) the nucleotide sequence of FIG. 6; and d) the nucleotide sequence of FIG.
 7. 22. A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in a genetically altered microorganism, modifies a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: a) expressing the mammalian gene in a genetically altered microorganism, thereby modifying the phenotypic alteration associated with the genetic alteration; b) contacting the genetically altered microorganism of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.
 23. A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) expressing the mammalian gene in genetically altered yeast cells in which the product encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration; b) contacting genetically altered yeast cells of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.
 24. The method of claim 23, wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.
 25. The method of claim 23, wherein the genetic alteration is a mutation in a gene encoding a RAS protein.
 26. The method of claim 25, wherein the genetic alteration is an activated RAS2^(va119) gene of S. cerevisiae.
 27. The method of claim 23 wherein the genetically altered yeast cells are genetically altered S. cerevisiae or genetically altered S. pombe.
 28. The method of claim 27 wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate, failure to mate and failure to sporulate.
 29. The method of claim 24 wherein the genetic alteration is a disruption of the PDE1 and a disruption of the PDE2 genes of S. cerevisiae.
 30. The method of claim 25 wherein the genetic alteration is a disruption of the ras1 gene of S. pombe or an activated allele of the ras1 gene of S. pombe.
 31. A method of identifying a chemical agent which alters activity of a protein encoded by a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: a) expressing the protein encoded by the mammalian gene in genetically altered yeast cells in which the protein encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration; b) obtaining from genetically altered yeast cells produced in step (a) protein encoded by the mammalian gene; c) combining protein encoded by the mammalian gene with a chemical agent to be assayed for its ability to alter activity of the protein encoded by the mammalian gene; and d) determining activity of the protein encoded by the mammalian gene in combination with the chemical agent. 